@article{barrangou_2024, title={AI and SynBio Meet CRISPR Heralding a New Genome Editing Era}, url={https://doi.org/10.1089/crispr.2024.0063}, DOI={10.1089/crispr.2024.0063}, journal={The CRISPR Journal}, author={Barrangou, Rodolphe}, year={2024}, month={Aug} }
 @article{roberts_spang_sanozky-dawes_nethery_barrangou_2024, title={Characterization of <i>Ligilactobacillus salivarius</i> CRISPR-Cas systems}, volume={7}, ISSN={["2379-5042"]}, url={https://doi.org/10.1128/msphere.00171-24}, DOI={10.1128/msphere.00171-24}, abstractNote={ABSTRACT Ligilactobacillus is a diverse genus among lactobacilli with phenotypes that reflect adaptation to various hosts. CRISPR-Cas systems are highly prevalent within lactobacilli, and Ligilactobacillus salivarius , the most abundant species of Ligilactobacillus , possesses both DNA- and RNA-targeting CRISPR-Cas systems. In this study, we explore the presence and functional properties of I-B, I-C, I-E, II-A, and III-A CRISPR-Cas systems in over 500 Ligilactobacillus genomes, emphasizing systems found in L. salivarius . We examined the I-E, II-A, and III-A CRISPR-Cas systems of two L. salivarius strains and observed occurrences of split cas genes and differences in CRISPR RNA maturation in native hosts. This prompted testing of the single Cas9 and multiprotein Cascade and Csm CRISPR-Cas effector complexes in a cell-free context to demonstrate the functionality of these systems. We also predicted self-targeting spacers within L. salivarius CRISPR-Cas systems and found that nearly a third of L. salivarius genomes possess unique self-targeting spacers that generally target elements other than prophages. With these two L. salivarius strains, we performed prophage induction coupled with RNA sequencing and discovered that the prophages residing within these strains are inducible and likely active elements, despite targeting by CRISPR-Cas systems. These findings deepen our comprehension of CRISPR-Cas systems in L. salivarius , further elucidating their relationship with associated prophages and providing a functional basis for the repurposing of these Cas effectors for bacterial manipulation. IMPORTANCE Ligilactobacillus salivarius is a diverse bacterial species widely used in the food and dietary supplement industries. In this study, we investigate the occurrence and diversity of their adaptive immune systems, CRISPR-Cas, in over 500 genomes. We establish their function and provide insights into their role in the interplay between the bacterial host and the predatory phages that infect them. Such findings expand our knowledge about these important CRISPR-Cas immune systems widespread across the bacterial tree of life and also provide a technical basis for the repurposing of these molecular machines for the development of molecular biology tools and the manipulation and engineering of bacteria and other life forms.}, journal={MSPHERE}, author={Roberts, Avery and Spang, Daniel and Sanozky-Dawes, Rosemary and Nethery, Matthew A. and Barrangou, Rodolphe}, editor={Ellermeier, Craig D.Editor}, year={2024}, month={Jul} }
 @article{davies_philippidis_barrangou_2024, title={Five Years of Progress in CRISPR Clinical Trials (2019-2024)}, volume={7}, ISSN={["2573-1602"]}, url={https://doi.org/10.1089/crispr.2024.0081}, DOI={10.1089/crispr.2024.0081}, abstractNote={In July 2019, Victoria Gray became the first patient with sickle cell disease to receive a CRISPR-based cell therapy as a volunteer in the exa-cel clinical trial, sponsored by Vertex Pharmaceuticals and CRISPR Therapeutics. Barely four years later, the ensuing therapy, branded as Casgevy, received approval from regulatory agencies in Europe, the United States, and the Middle East, ushering in a new era of CRISPR-based medicines. During this period, scores of other clinical trials have been launched, including many actively recruiting patients across phase 1, phase 2, and phase 3 clinical trials around the world. In this brief Perspective, we collate the latest information on therapeutic clinical trials featuring CRISPR, base and prime editing, across a range of both}, number={5}, journal={CRISPR JOURNAL}, author={Davies, Kevin and Philippidis, Alex and Barrangou, Rodolphe}, year={2024}, month={Oct}, pages={227–230} }
 @article{barrangou_2024, title={Surveying the State of CRISPR and Gene Editing}, volume={7}, ISSN={["2573-1602"]}, url={https://doi.org/10.1089/crispr.2024.0045}, DOI={10.1089/crispr.2024.0045}, number={3}, journal={CRISPR JOURNAL}, author={Barrangou, Rodolphe}, year={2024}, month={Jun}, pages={133–134} }
 @article{qaim_barrangou_ronald_2024, title={Sustainability of animal-sourced foods and plant-based alternatives}, url={https://doi.org/10.1073/pnas.2400495121}, DOI={10.1073/pnas.2400495121}, journal={Proceedings of the National Academy of Sciences}, author={Qaim, Matin and Barrangou, Rodolphe and Ronald, Pamela C.}, year={2024}, month={Dec} }
 @article{barrangou_2024, title={‘Tis the Season: CRISPR Products All Around}, url={https://doi.org/10.1089/crispr.2024.0094}, DOI={10.1089/crispr.2024.0094}, journal={The CRISPR Journal}, author={Barrangou, Rodolphe}, year={2024}, month={Dec} }
 @article{gilfillan_vilander_pan_goh_o'flaherty_feng_fox_lang_greenberg_abdo_et al._2023, title={<i>Lactobacillus acidophilus</i> Expressing Murine Rotavirus VP8 and Mucosal Adjuvants Induce Virus-Specific Immune Responses}, volume={11}, ISSN={["2076-393X"]}, url={https://www.mdpi.com/2076-393X/11/12/1774}, DOI={10.3390/vaccines11121774}, abstractNote={Rotavirus diarrhea-associated illness remains a major cause of global death in children under five, attributable in part to discrepancies in vaccine performance between high- and low-middle-income countries. Next-generation probiotic vaccines could help bridge this efficacy gap. We developed a novel recombinant Lactobacillus acidophilus (rLA) vaccine expressing rotavirus antigens of the VP8* domain from the rotavirus EDIM VP4 capsid protein along with the adjuvants FimH and FliC. The upp-based counterselective gene-replacement system was used to chromosomally integrate FimH, VP8Pep (10 amino acid epitope), and VP8-1 (206 amino acid protein) into the L. acidophilus genome, with FliC expressed from a plasmid. VP8 antigen and adjuvant expression were confirmed by flow cytometry and Western blot. Rotavirus naïve adult BALB/cJ mice were orally immunized followed by murine rotavirus strain ECWT viral challenge. Antirotavirus serum IgG and antigen-specific antibody-secreting cell responses were detected in rLA-vaccinated mice. A day after the oral rotavirus challenge, fecal antigen shedding was significantly decreased in the rLA group. These results indicate that novel rLA constructs expressing VP8 can be successfully constructed and used to generate modest homotypic protection from rotavirus challenge in an adult murine model, indicating the potential for a probiotic next-generation vaccine construct against human rotavirus.}, number={12}, journal={VACCINES}, author={Gilfillan, Darby and Vilander, Allison C. and Pan, Meichen and Goh, Yong Jun and O'Flaherty, Sarah and Feng, Ningguo and Fox, Bridget E. and Lang, Callie and Greenberg, Harry B. and Abdo, Zaid and et al.}, year={2023}, month={Dec} }
 @article{foley_walker_stewart_o'flaherty_gentry_patel_beaty_allen_pan_simpson_et al._2023, title={Bile salt hydrolases shape the bile acid landscape and restrict Clostridioides difficile growth in the murine gut}, volume={3}, ISSN={["2058-5276"]}, DOI={10.1038/s41564-023-01337-7}, abstractNote={AbstractBile acids (BAs) mediate the crosstalk between human and microbial cells and influence diseases including Clostridioides difficile infection (CDI). While bile salt hydrolases (BSHs) shape the BA pool by deconjugating conjugated BAs, the basis for their substrate selectivity and impact on C. difficile remain elusive. Here we survey the diversity of BSHs in the gut commensals Lactobacillaceae, which are commonly used as probiotics, and other members of the human gut microbiome. We structurally pinpoint a loop that predicts BSH preferences for either glycine or taurine substrates. BSHs with varying specificities were shown to restrict C. difficile spore germination and growth in vitro and colonization in pre-clinical in vivo models of CDI. Furthermore, BSHs reshape the pool of microbial conjugated bile acids (MCBAs) in the murine gut, and these MCBAs can further restrict C. difficile virulence in vitro. The recognition of conjugated BAs by BSHs defines the resulting BA pool, including the expansive MCBAs. This work provides insights into the structural basis of BSH mechanisms that shape the BA landscape and promote colonization resistance against C. difficile.}, journal={NATURE MICROBIOLOGY}, author={Foley, Matthew H. and Walker, Morgan E. and Stewart, Allison K. and O'Flaherty, Sarah and Gentry, Emily C. and Patel, Shakshi and Beaty, Violet V. and Allen, Garrison and Pan, Meichen and Simpson, Joshua B. and et al.}, year={2023}, month={Mar} }
 @article{barrangou_2023, title={CRISPR Technology and its Many Applications with Select Examples Related to Animal Agriculture}, volume={101}, ISSN={["1525-3163"]}, DOI={10.1093/jas/skad068.001}, abstractNote={Abstract
               Dr. Barrangou’s research focuses on the biology and genetics of CRISPR-Cas immune systems in bacteria. Using microbiology, molecular biology and genomics approaches, he investigates the use of CRISPR-Cas systems to generate novel tools for the manipulation of industrially relevant organisms for food and biotechnological applications. Dr. Barrangou talk will be an update on CRISPR technology and its many applications with select examples related to animal agriculture.}, journal={JOURNAL OF ANIMAL SCIENCE}, author={Barrangou, Rodolphe}, year={2023}, month={May} }
 @article{adler_trinidad_bellieny-rabelo_zhang_karp_skopintsev_thornton_weissman_yoon_chen_et al._2023, title={CasPEDIA Database: a functional classification system for class 2 CRISPR-Cas enzymes}, volume={10}, ISSN={["1362-4962"]}, url={https://doi.org/10.1093/nar/gkad890}, DOI={10.1093/nar/gkad890}, abstractNote={Abstract
               CRISPR-Cas enzymes enable RNA-guided bacterial immunity and are widely used for biotechnological applications including genome editing. In particular, the Class 2 CRISPR-associated enzymes (Cas9, Cas12 and Cas13 families), have been deployed for numerous research, clinical and agricultural applications. However, the immense genetic and biochemical diversity of these proteins in the public domain poses a barrier for researchers seeking to leverage their activities. We present CasPEDIA (http://caspedia.org), the Cas Protein Effector Database of Information and Assessment, a curated encyclopedia that integrates enzymatic classification for hundreds of different Cas enzymes across 27 phylogenetic groups spanning the Cas9, Cas12 and Cas13 families, as well as evolutionarily related IscB and TnpB proteins. All enzymes in CasPEDIA were annotated with a standard workflow based on their primary nuclease activity, target requirements and guide-RNA design constraints. Our functional classification scheme, CasID, is described alongside current phylogenetic classification, allowing users to search related orthologs by enzymatic function and sequence similarity. CasPEDIA is a comprehensive data portal that summarizes and contextualizes enzymatic properties of widely used Cas enzymes, equipping users with valuable resources to foster biotechnological development. CasPEDIA complements phylogenetic Cas nomenclature and enables researchers to leverage the multi-faceted nucleic-acid targeting rules of diverse Class 2 Cas enzymes.}, journal={NUCLEIC ACIDS RESEARCH}, author={Adler, Benjamin A. and Trinidad, Marena I and Bellieny-Rabelo, Daniel and Zhang, Elaine and Karp, Hannah M. and Skopintsev, Petr and Thornton, Brittney W. and Weissman, Rachel F. and Yoon, Peter H. and Chen, Linxing and et al.}, year={2023}, month={Oct} }
 @article{page_perez-diaz_pan_barrangou_2023, title={Genome-Wide Comparative Analysis of Lactiplantibacillus pentosus Isolates Autochthonous to Cucumber Fermentation Reveals Subclades of Divergent Ancestry}, volume={12}, ISSN={["2304-8158"]}, url={https://doi.org/10.3390/foods12132455}, DOI={10.3390/foods12132455}, abstractNote={Lactiplantibacillus pentosus, commonly isolated from commercial cucumber fermentation, is a promising candidate for starter culture formulation due to its ability to achieve complete sugar utilization to an end pH of 3.3. In this study, we conducted a comparative genomic analysis encompassing 24 L. pentosus and 3 Lactiplantibacillus plantarum isolates autochthonous to commercial cucumber fermentation and 47 lactobacillales reference genomes to determine species specificity and provide insights into niche adaptation. Results showed that metrics such as average nucleotide identity score, emulated Rep-PCR-(GTG)5, computed multi-locus sequence typing (MLST), and multiple open reading frame (ORF)-based phylogenetic trees can robustly and consistently distinguish the two closely related species. Phylogenetic trees based on the alignment of 587 common ORFs separated the L. pentosus autochthonous cucumber isolates from olive fermentation isolates into clade A and B, respectively. The L. pentosus autochthonous clade partitions into subclades A.I, A.II, and A.III, suggesting substantial intraspecies diversity in the cucumber fermentation habitat. The hypervariable sequences within CRISPR arrays revealed recent evolutionary history, which aligns with the L. pentosus subclades identified in the phylogenetic trees constructed. While L. plantarum autochthonous to cucumber fermentation only encode for Type II-A CRISPR arrays, autochthonous L. pentosus clade B codes for Type I-E and L. pentosus clade A hosts both types of arrays. L. pentosus 7.8.2, for which phylogeny could not be defined using the varied methods employed, was found to uniquely encode for four distinct Type I-E CRISPR arrays and a Type II-A array. Prophage sequences in varied isolates evidence the presence of adaptive immunity in the candidate starter cultures isolated from vegetable fermentation as observed in dairy counterparts. This study provides insight into the genomic features of industrial Lactiplantibacillus species, the level of species differentiation in a vegetable fermentation habitat, and diversity profile of relevance in the selection of functional starter cultures.}, number={13}, journal={FOODS}, author={Page, Clinton A. and Perez-Diaz, Ilenys M. and Pan, Meichen and Barrangou, Rodolphe}, year={2023}, month={Jul} }
 @article{o'flaherty_cobian_barrangou_2023, title={Impact of Pomegranate on Probiotic Growth, Viability, Transcriptome and Metabolism}, volume={11}, ISSN={["2076-2607"]}, url={https://doi.org/10.3390/microorganisms11020404}, DOI={10.3390/microorganisms11020404}, abstractNote={Despite rising interest in understanding intestinal bacterial survival in situ, relatively little attention has been devoted to deciphering the interaction between bacteria and functional food ingredients. Here, we examined the interplay between diverse beneficial Lactobacillaceae species and a pomegranate (POM) extract and determined the impact of this functional ingredient on bacterial growth, cell survival, transcription and target metabolite genesis. Three commercially available probiotic strains (Lactobacillus acidophilus NCFM, Lacticaseibacillus rhamnosus GG and Lactiplantibacillus plantarum Lp-115) were used in growth assays and flow cytometry analysis, indicating differential responses to the presence of POM extract across the three strains. The inclusion of POM extract in the growth medium had the greatest impact on L. acidophilus cell counts. LIVE/DEAD staining determined significantly fewer dead cells when L. acidophilus was grown with POM extract compared to the control with no POM (1.23% versus 7.23%). Whole-transcriptome analysis following exposure to POM extract showed markedly different global transcriptome responses, with 15.88% of the L. acidophilus transcriptome, 19.32% of the L. rhamnosus transcriptome and only 2.37% of the L. plantarum transcriptome differentially expressed. We also noted strain-dependent metabolite concentrations in the medium with POM extract compared to the control medium for punicalagin, ellagic acid and gallic acid. Overall, the results show that POM extract triggers species-specific responses by probiotic strains and substantiates the rising interest in using POM as a prebiotic compound.}, number={2}, journal={MICROORGANISMS}, author={O'Flaherty, Sarah and Cobian, Natalia and Barrangou, Rodolphe}, year={2023}, month={Feb} }
 @article{raftopoulou_barrangou_2023, title={Mining microbial organisms to discover and characterize novel CRISPR-Cas systems}, volume={27}, ISSN={["2468-4511"]}, url={https://doi.org/10.1016/j.cobme.2023.100469}, DOI={10.1016/j.cobme.2023.100469}, abstractNote={The need for new genome manipulation tools is leading the way for the continued discovery of novel clustered regularly interspaced short palindromic repeats— CRISPR associated sequences (CRISPR-Cas) systems. Researchers have been analyzing the genomes of prokaryotes and more recently metagenomic sequencing data to find novel and diverse CRISPR-Cas systems and their associated genome editing effectors. In this review, we provide an overview of in silico, in vitro, and in vivo analyses performed to characterize key elements of CRISPR-Cas systems, encompassing the CRISPR array, Cas proteins, guide ribonucleic acid (RNAs), and protospacer-adjacent motif (PAM) which defines targeting. We also highlight subsequent in vitro and in vivo assays employed to validate CRISPR function and Cas effector activity in the context of genome editing in various cellular contexts.}, journal={CURRENT OPINION IN BIOMEDICAL ENGINEERING}, author={Raftopoulou, Ourania and Barrangou, Rodolphe}, year={2023}, month={Sep} }
 @article{sulis_jiang_yang_marques_matthews_miller_lan_cofre-vega_liu_sun_et al._2023, title={Multiplex CRISPR editing of wood for sustainable fiber production}, volume={381}, ISSN={["1095-9203"]}, url={http://europepmc.org/abstract/med/37440632}, DOI={10.1126/science.add4514}, abstractNote={The domestication of forest trees for a more sustainable fiber bioeconomy has long been hindered by the complexity and plasticity of lignin, a biopolymer in wood that is recalcitrant to chemical and enzymatic degradation. Here, we show that multiplex CRISPR editing enables precise woody feedstock design for combinatorial improvement of lignin composition and wood properties. By assessing every possible combination of 69,123 multigenic editing strategies for 21 lignin biosynthesis genes, we deduced seven different genome editing strategies targeting the concurrent alteration of up to six genes and produced 174 edited poplar variants. CRISPR editing increased the wood carbohydrate-to-lignin ratio up to 228% that of wild type, leading to more-efficient fiber pulping. The edited wood alleviates a major fiber-production bottleneck regardless of changes in tree growth rate and could bring unprecedented operational efficiencies, bioeconomic opportunities, and environmental benefits.}, number={6654}, journal={SCIENCE}, author={Sulis, Daniel B. and Jiang, Xiao and Yang, Chenmin and Marques, Barbara M. and Matthews, Megan L. and Miller, Zachary and Lan, Kai and Cofre-Vega, Carlos and Liu, Baoguang and Sun, Runkun and et al.}, year={2023}, month={Jul}, pages={216-+} }
 @article{pyhtila_kasowitz_leeson_barrangou_2023, title={The Expanding Dissemination and Distribution Patterns of Diverse CRISPR Plasmids by Addgene}, volume={11}, ISSN={["2573-1602"]}, url={https://doi.org/10.1089/crispr.2023.0059}, DOI={10.1089/crispr.2023.0059}, abstractNote={CRISPR-based technologies have rapidly enabled the democratization of genome editing in academic institutions through distribution by Addgene over the past decade. Recently, several distribution milestones have been reached, with a collection of >15,000 plasmids deposited by >1,000 laboratories spanning ∼40 countries now shipped 300,000 times to ∼5,000 organizations traversing ∼100 countries. Yet, both deposits of and requests for CRISPR plasmids continue to rise for this disruptive technology. Distribution patterns revealed robust demand for three distinct classes of CRISPR effectors, namely nucleases (e.g., Cas9 and Cas12), modulators (deactivated CRISPR nucleases fused to transcriptional regulators and epigenome modifiers), and chimeric effectors (Cas proteins fused to enzymes carrying out other activities such as deamination, reverse transcription, transposition, and integration). Yearly deposits over the past decade are requested in near-even proportions, reflecting continuous technological development and requests for novel constructs. Though it is unclear whether the slowing rate of requests is inherent to a pandemic operational lag or a transition from emerging to mature technology, it is noteworthy that the relative proportion of requests from plasmids deposited in the previous year remains stable, suggesting robust development of novel tools concurrent with continued adoption of editing, base editing, prime editing, and more. Predictably, most requested plasmids are designed for mammalian genome manipulation, presumably for medical research and human health pursuits, reflecting investments in therapeutic applications. Concurrently, requests for plant and microbial constructs are on the rise, especially in regions of the world more reliant on local agricultural inputs and focused on food and feed applications, illustrating continued diversification of genome editing applications.}, journal={CRISPR JOURNAL}, author={Pyhtila, Brook and Kasowitz, Seth and Leeson, Rachel and Barrangou, Rodolphe}, year={2023}, month={Nov} }
 @article{adler_hessler_cress_lahiri_mutalik_barrangou_banfield_doudna_2022, title={Broad-spectrum CRISPR-Cas13a enables efficient phage genome editing}, volume={10}, ISSN={["2058-5276"]}, url={https://doi.org/10.1038/s41564-022-01258-x}, DOI={10.1038/s41564-022-01258-x}, abstractNote={AbstractCRISPR-Cas13 proteins are RNA-guided RNA nucleases that defend against incoming RNA and DNA phages by binding to complementary target phage transcripts followed by general, non-specific RNA degradation. Here we analysed the defensive capabilities of LbuCas13a from Leptotrichia buccalis and found it to have robust antiviral activity unaffected by target phage gene essentiality, gene expression timing or target sequence location. Furthermore, we find LbuCas13a antiviral activity to be broadly effective against a wide range of phages by challenging LbuCas13a against nine E. coli phages from diverse phylogenetic groups. Leveraging the versatility and potency enabled by LbuCas13a targeting, we applied LbuCas13a towards broad-spectrum phage editing. Using a two-step phage-editing and enrichment method, we achieved seven markerless genome edits in three diverse phages with 100% efficiency, including edits as large as multi-gene deletions and as small as replacing a single codon. Cas13a can be applied as a generalizable tool for editing the most abundant and diverse biological entities on Earth.}, journal={NATURE MICROBIOLOGY}, author={Adler, Benjamin A. and Hessler, Tomas and Cress, Brady F. and Lahiri, Arushi and Mutalik, Vivek K. and Barrangou, Rodolphe and Banfield, Jillian and Doudna, Jennifer A.}, year={2022}, month={Oct} }
 @article{nethery_hidalgo-cantabrana_roberts_barrangou_2022, title={CRISPR-based engineering of phages for in situ bacterial base editing}, volume={119}, ISSN={["1091-6490"]}, url={https://doi.org/10.1073/pnas.2206744119}, DOI={10.1073/pnas.2206744119}, abstractNote={
            Investigation of microbial gene function is essential to the elucidation of ecological roles and complex genetic interactions that take place in microbial communities. While microbiome studies have increased in prevalence, the lack of viable in situ editing strategies impedes experimental progress, rendering genetic knowledge and manipulation of microbial communities largely inaccessible. Here, we demonstrate the utility of phage-delivered CRISPR-Cas payloads to perform targeted genetic manipulation within a community context, deploying a fabricated ecosystem (EcoFAB) as an analog for the soil microbiome. First, we detail the engineering of two classical phages for community editing using recombination to replace nonessential genes through Cas9-based selection. We show efficient engineering of T7, then demonstrate the expression of antibiotic resistance and fluorescent genes from an engineered λ prophage within an
            Escherichia coli
            host. Next, we modify λ to express an APOBEC-1-based cytosine base editor (CBE), which we leverage to perform C-to-T point mutations guided by a modified Cas9 containing only a single active nucleolytic domain (nCas9). We strategically introduce these base substitutions to create premature stop codons in-frame, inactivating both chromosomal (
            lacZ
            ) and plasmid-encoded genes (mCherry and ampicillin resistance) without perturbation of the surrounding genomic regions. Furthermore, using a multigenera synthetic soil community, we employ phage-assisted base editing to induce host-specific phenotypic alterations in a community context both in vitro and within the EcoFAB, observing editing efficiencies from 10 to 28% across the bacterial population. The concurrent use of a synthetic microbial community, soil matrix, and EcoFAB device provides a controlled and reproducible model to more closely approximate in situ editing of the soil microbiome.
          }, number={46}, journal={PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA}, author={Nethery, Matthew A. and Hidalgo-Cantabrana, Claudio and Roberts, Avery and Barrangou, Rodolphe}, year={2022}, month={Nov} }
 @article{monte_nethery_berman_keelara_lincopan_fedorka-cray_barrangou_landgraf_2022, title={Clustered Regularly Interspaced Short Palindromic Repeats Genotyping of Multidrug-Resistant Salmonella Heidelberg Strains Isolated From the Poultry Production Chain Across Brazil}, volume={13}, ISSN={["1664-302X"]}, DOI={10.3389/fmicb.2022.867278}, abstractNote={Salmonella enterica subsp. enterica serovar Heidelberg has been associated with a broad host range, such as poultry, dairy calves, swine, wild birds, environment, and humans. The continuous evolution of S. Heidelberg raises a public health concern since there is a global dispersal of lineages harboring a wide resistome and virulome on a global scale. Here, we characterized the resistome, phylogenetic structure and clustered regularly interspaced short palindromic repeats (CRISPR) array composition of 81 S. Heidelberg strains isolated from broiler farms (n = 16), transport and lairage (n = 5), slaughterhouse (n = 22), and retail market (n = 38) of the poultry production chain in Brazil, between 2015 and 2016 using high-resolution approaches including whole-genome sequencing (WGS) and WGS-derived CRISPR genotyping. More than 91% of the S. Heidelberg strains were multidrug-resistant. The total antimicrobial resistance (AMR) gene abundances did not vary significantly across regions and sources suggesting the widespread distribution of antibiotic-resistant strains from farm to market. The highest AMR gene abundance was observed for fosA7, aac(6′)-Iaa, sul2, tet(A), gyrA, and parC for 100% of the isolates, followed by 88.8% for blaCMY–2. The β-lactam resistance was essentially driven by the presence of the plasmid-mediated AmpC (pAmpC) blaCMY–2 gene, given the isolates which did not carry this gene were susceptible to cefoxitin (FOX). Most S. Heidelberg strains were classified within international lineages, which were phylogenetically nested with Salmonella strains from European countries; while CRISPR genotyping analysis revealed that the spacer content was overall highly conserved, but distributed into 13 distinct groups. In summary, our findings underscore the potential role of S. Heidelberg as a key pathogen disseminated from farm to fork in Brazil and reinforce the importance of CRISPR-based genotyping for salmonellae. Hence, we emphasized the need for continuous mitigation programs to monitor the dissemination of this high-priority pathogen.}, journal={FRONTIERS IN MICROBIOLOGY}, author={Monte, Daniel F. M. and Nethery, Matthew A. and Berman, Hanna and Keelara, Shivaramu and Lincopan, Nilton and Fedorka-Cray, Paula J. and Barrangou, Rodolphe and Landgraf, Mariza}, year={2022}, month={Jun} }
 @article{roberts_nethery_barrangou_2022, title={Functional characterization of diverse type I-F CRISPR-associated transposons}, volume={11}, ISSN={["1362-4962"]}, url={https://doi.org/10.1093/nar/gkac985}, DOI={10.1093/nar/gkac985}, abstractNote={Abstract
               CRISPR-Cas systems generally provide adaptive immunity in prokaryotes through RNA-guided degradation of foreign genetic elements like bacteriophages and plasmids. Recently, however, transposon-encoded and nuclease-deficient CRISPR-Cas systems were characterized and shown to be co-opted by Tn7-like transposons for CRISPR RNA-guided DNA transposition. As a genome engineering tool, these CRISPR-Cas systems and their associated transposon proteins can be deployed for programmable, site-specific integration of sizable cargo DNA, circumventing the need for DNA cleavage and homology-directed repair involving endogenous repair machinery. Here, we selected a diverse set of type I-F3 CRISPR-associated transposon systems derived from Gammaproteobacteria, predicted all components essential for transposition activity, and deployed them for functionality testing within Escherichia coli. Our results demonstrate that these systems possess a significant range of integration efficiencies with regards to temperature, transposon size, and flexible PAM requirements. Additionally, our findings support the categorization of these systems into functional compatibility groups for efficient and orthogonal RNA-guided DNA integration. This work expands the CRISPR-based toolbox with new CRISPR RNA-guided DNA integrases that can be applied to complex and extensive genome engineering efforts.}, journal={NUCLEIC ACIDS RESEARCH}, author={Roberts, Avery and Nethery, Matthew A. and Barrangou, Rodolphe}, year={2022}, month={Nov} }
 @article{pan_morovic_hidalgo-cantabrana_roberts_walden_goh_barrangou_2022, title={Genomic and epigenetic landscapes drive CRISPR-based genome editing in Bifidobacterium}, volume={119}, ISSN={["1091-6490"]}, url={https://doi.org/10.1073/pnas.2205068119}, DOI={10.1073/pnas.2205068119}, abstractNote={Bifidobacteriumis a commensal bacterial genus ubiquitous in the human gastrointestinal tract, which is associated with a range of health benefits. The advent of CRISPR-based genome editing technologies provides opportunities to investigate the genetics of important bacteria and transcend the lack of genetic tools in bifidobacteria to study the basis for their health-promoting attributes. Here, we repurpose the endogenous type I-G CRISPR-Cas system and adopt an exogenous CRISPR base editor for genome engineering inB. animalissubsp.lactis,demonstrating that both genomic and epigenetic contexts drive editing outcomes across strains. We reprogrammed the endogenous type I-G system to screen for naturally occurring large deletions up to 27 kb and to generate a 500-bp deletion intetWto abolish tetracycline resistance. A CRISPR-cytosine base editor was optimized to install C•G-to-T•A amber mutations to resensitize multipleB. lactisstrains to tetracycline. Remarkably, we uncovered epigenetic patterns that are distributed unevenly amongB. lactisstrains, despite their genomic homogeneity, that may contribute to editing efficiency variability. Insights were also expanded toBifidobacterium longumsubsp.infantisto emphasize the broad relevance of these findings. This study highlights the need to develop individualized CRISPR-based genome engineering approaches for distinct bacterial strains and opens avenues for engineering of next generation probiotics.}, number={30}, journal={PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA}, author={Pan, Meichen and Morovic, Wesley and Hidalgo-Cantabrana, Claudio and Roberts, Avery and Walden, Kimberly K. O. and Goh, Yong Jun and Barrangou, Rodolphe}, year={2022}, month={Jul} }
 @article{chamberlain_o'flaherty_cobian_barrangou_2022, title={Metabolomic Analysis of Lactobacillus acidophilus, L. gasseri, L. crispatus, and Lacticaseibacillus rhamnosus Strains in the Presence of Pomegranate Extract}, volume={13}, ISSN={["1664-302X"]}, DOI={10.3389/fmicb.2022.863228}, abstractNote={Lactobacillus species are prominent inhabitants of the human gastrointestinal tract that contribute to maintaining a balanced microbial environment that positively influences host health. These bacterial populations can be altered through use of probiotic supplements or via dietary changes which in turn affect the host health. Utilizing polyphenolic compounds to selectively stimulate the growth of commensal bacteria can have a positive effect on the host through the production of numerous metabolites that are biologically active. Four Lactobacillus strains were grown in the presence of pomegranate (POM) extract. Two strains, namely, L. acidophilus NCFM and L. rhamnosus GG, are commonly used probiotics, while the other two strains, namely, L. crispatus NCK1351 and L. gasseri NCK1342, exhibit probiotic potential. To compare and contrast the impact of POM on the strains' metabolic capacity, we investigated the growth of the strains with and without the presence of POM and identified their carbohydrate utilization and enzyme activity profiles. To further investigate the differences between strains, an untargeted metabolomic approach was utilized to quantitatively and qualitatively define the metabolite profiles of these strains. Several metabolites were produced significantly and/or exclusively in some of the strains, including mevalonate, glutamine, 5-aminoimidazole-4-carboxamide, phenyllactate, and fumarate. The production of numerous discrete compounds illustrates the unique characteristics of and diversity between strains. Unraveling these differences is essential to understand the probiotic function and help inform strain selection for commercial product formulation.}, journal={FRONTIERS IN MICROBIOLOGY}, author={Chamberlain, MaryClaire and O'Flaherty, Sarah and Cobian, Natalia and Barrangou, Rodolphe}, year={2022}, month={May} }
 @article{barrangou_2022, title={Next-Generation Foods and CRISPR Engineering}, url={https://doi.org/10.52750/106366}, DOI={10.52750/106366}, abstractNote={The advent of CRISPR-based technologies has revolutionized our ability to manipulate the genomes of virtually every entity across the tree of life.Besides the tremendous progress in the medical applications of CRISPR technologies for gene therapies and cell engineering in the clinic, there are tremendous opportunities to exploit genome editing for food and agriculture.Indeed, breeding of crops and livestock can address grand challenges for our food supply chain and revolutionize agriculture at a time when resources are scarce and sustainability is crucial.Rodolphe Barrangou, Ph.D., discusses how genome editing is opening new avenues for a more sustainable agriculture.Barrangou is the T.R. Klaenhammer Distinguished professor at NC State.He is focusing on the characterization of CRISPR-Cas systems, and their applications in bacteria.Barrangou spent nine years in research and development, and mergers and acquisitions at Danisco and DuPont.}, author={Barrangou, Rodolphe}, year={2022}, month={Aug} }
 @article{adler_hessler_cress_mutalik_barrangou_banfield_doudna_2022, title={RNA-targeting CRISPR-Cas13 Provides Broad-spectrum Phage Immunity}, volume={3}, url={https://doi.org/10.1101/2022.03.25.485874}, DOI={10.1101/2022.03.25.485874}, abstractNote={AbstractCRISPR-Cas13 proteins are RNA-guided RNA nucleases that defend against invasive phages through general, non-specific RNA degradation upon complementary target transcript binding. Despite being RNA nucleases, Cas13 effectors are capable of inhibiting the infection of dsDNA phages but have only been investigated across a relatively small sampling of phage diversity. Here, we employ a systematic, phage-centric approach to determine the anti-phage capacity of Cas13 and find LbuCas13a to be a remarkably potent phage inhibitor. LbuCas13a confers robust, consistent antiviral activity regardless of gene essentiality, gene expression timing or target sequence location. Furthermore, after challenging LbuCas13a with eight diverse E. coli phages distributed across E. coli phage phylogenetic groups, we find no apparent phage-encoded limits to its potent antiviral activity. In contrast to other Class 2 CRISPR-Cas proteins, these results suggest that DNA phages are generally vulnerable to Cas13a targeting. Leveraging this effective anti-phage activity, LbuCas13a can be used seamlessly as a counter-selection agent for broad-spectrum phage editing. Using a two-step phage editing and enrichment approach, we show that LbuCas13a enables markerless genome edits in phages with exceptionally high efficiency and precision, including edits as small as a single codon. By taking advantage of the broad vulnerability of RNA during viral infection, Cas13a enables a generalizable strategy for editing the most abundant and diverse biological entities on Earth.}, publisher={Cold Spring Harbor Laboratory}, author={Adler, Benjamin A. and Hessler, Tomas and Cress, Brady F and Mutalik, Vivek K. and Barrangou, Rodolphe and Banfield, Jillian and Doudna, Jennifer A}, year={2022}, month={Mar} }
 @article{barrangou_marraffini_2022, title={Turning CRISPR on with antibiotics}, volume={30}, ISSN={["1934-6069"]}, DOI={10.1016/j.chom.2021.12.013}, abstractNote={CRISPR-Cas systems have the ability to integrate invasive DNA sequences to build adaptive immunity in bacteria. In this issue Dimitriu et al. show bacteriostatic antibiotics prompt CRISPR acquisition events, illustrating how environmental conditions affect complex dynamics between host and virus and the corresponding biological and genetic arms race.}, number={1}, journal={CELL HOST & MICROBE}, author={Barrangou, Rodolphe and Marraffini, Luciano A.}, year={2022}, month={Jan}, pages={12–14} }
 @article{kuiken_barrangou_grieger_2021, title={(Broken) Promises of Sustainable Food and Agriculture through New Biotechnologies: The CRISPR Case}, volume={4}, ISSN={["2573-1602"]}, DOI={10.1089/crispr.2020.0098}, abstractNote={In recent years, the development of diverse CRISPR-based technologies has revolutionized genome manipulation and enabled a broad scientific community in industry, academia, and beyond to redefine research and development for biotechnology products encompassing food, agriculture, and medicine. CRISPR-based genome editing affords tremendous opportunities in agriculture for the breeding of crops and livestock across the food supply chain that could benefit larger portions of the population compared to CRISPR applications in medicine, for example by helping to feed a growing global population, reach sustainability goals, and possibly mitigate the effects of climate change. These promises come alongside concerns of risks and adverse impacts associated with CRISPR-based genome editing and concerns that governance systems that are ill equipped or not well suited to evaluate these risks. The international community will continue to gather, in multiple venues, in the coming years to discuss these concerns. At the same time, responsible research and innovation paradigms also promise to evaluate the risks and benefits better while incorporating broad stakeholder engagement across the research and development process. The CRISPR community therefore must actively engage with these international deliberations, society, and national governance systems that have promised to build better agricultural systems and provide better food products to achieve equitable outcomes while protecting the environment. Without this active engagement, the promises discussed in this paper are sure to be broken.}, number={1}, journal={CRISPR JOURNAL}, author={Kuiken, Todd and Barrangou, Rodolphe and Grieger, Khara}, year={2021}, month={Feb}, pages={25–31} }
 @article{nethery_korvink_makarova_wolf_v. koonin_barrangou_2021, title={CRISPRclassify: Repeat-Based Classification of CRISPR Loci}, volume={4}, ISSN={["2573-1602"]}, DOI={10.1089/crispr.2021.0021}, abstractNote={Detection and classification of CRISPR-Cas systems in metagenomic data have become increasingly prevalent in recent years due to their potential for diverse applications in genome editing. Traditionally, CRISPR-Cas systems are classified through reference-based identification of proximate cas genes. Here, we present a machine learning approach for the detection and classification of CRISPR loci using repeat sequences in a cas-independent context, enabling identification of unclassified loci missed by traditional cas-based approaches. Using biological attributes of the CRISPR repeat, the core element in CRISPR arrays, and leveraging methods from natural language processing, we developed a machine learning model capable of accurate classification of CRISPR loci in an extensive set of metagenomes, resulting in an F1 measure of 0.82 across all predictions and an F1 measure of 0.97 when limiting to classifications with probabilities >0.85. Furthermore, assessing performance on novel repeats yielded an F1 measure of 0.96. Although the performance of cas-based identification will exceed that of a repeat-based approach in many cases, CRISPRclassify provides an efficient approach to classification of CRISPR loci for cases in which cas gene information is unavailable, such as metagenomes and fragmented genome assemblies.}, number={4}, journal={CRISPR JOURNAL}, author={Nethery, Matthew A. and Korvink, Michael and Makarova, Kira S. and Wolf, Yuri I. and V. Koonin, Eugene and Barrangou, Rodolphe}, year={2021}, month={Aug}, pages={558–574} }
 @article{cobian_garlet_hidalgo-cantabrana_barrangou_2021, title={Comparative Genomic Analyses and CRISPR-Cas Characterization of Cutibacterium acnes Provide Insights Into Genetic Diversity and Typing Applications}, volume={12}, ISSN={["1664-302X"]}, DOI={10.3389/fmicb.2021.758749}, abstractNote={Cutibacterium acnes is an important member of the human skin microbiome and plays a critical role in skin health and disease. C. acnes encompasses different phylotypes that have been found to be associated with different skin phenotypes, suggesting a genetic basis for their impact on skin health. Here, we present a comprehensive comparative analysis of 255 C. acnes genomes to provide insights into the species genetic diversity and identify unique features that define various phylotypes. Results revealed a relatively small and open pan genome (6,240 genes) with a large core genome (1,194 genes), and three distinct phylogenetic clades, with multiple robust sub-clades. Furthermore, we identified several unique gene families driving differences between distinct C. acnes clades. Carbohydrate transporters, stress response mechanisms and potential virulence factors, potentially involved in competitive growth and host colonization, were detected in type I strains, which are presumably responsible for acne. Diverse type I-E CRISPR-Cas systems and prophage sequences were detected in select clades, providing insights into strain divergence and adaptive differentiation. Collectively, these results enable to elucidate the fundamental differences among C. acnes phylotypes, characterize genetic elements that potentially contribute to type I-associated dominance and disease, and other key factors that drive the differentiation among clades and sub-clades. These results enable the use of comparative genomics analyses as a robust method to differentiate among the C. acnes genotypes present in the skin microbiome, opening new avenues for the development of biotherapeutics to manipulate the skin microbiota.}, journal={FRONTIERS IN MICROBIOLOGY}, author={Cobian, Natalia and Garlet, Allison and Hidalgo-Cantabrana, Claudio and Barrangou, Rodolphe}, year={2021}, month={Nov} }
 @article{goh_barrangou_klaenhammer_2021, title={In Vivo Transcriptome of Lactobacillus acidophilus and Colonization Impact on Murine Host Intestinal Gene Expression}, volume={12}, ISSN={["2150-7511"]}, url={https://doi.org/10.1128/mBio.03399-20}, DOI={10.1128/mBio.03399-20}, abstractNote={
            To date, our basis for comprehending the probiotic mechanisms of
            Lactobacillus acidophilus
            , one of the most widely consumed probiotic microbes, was largely limited to
            in vitro
            functional genomic studies. Using a germfree murine colonization model,
            in vivo
            -based transcriptional studies provided the first view of how
            L. acidophilus
            survives in the mammalian gut environment, including gene expression patterns linked to survival, efficient nutrient acquisition, stress adaptation, and host interactions.
          }, number={1}, journal={MBIO}, publisher={American Society for Microbiology}, author={Goh, Yong Jun and Barrangou, Rodolphe and Klaenhammer, Todd R.}, editor={Huffnagle, Gary B.Editor}, year={2021} }
 @article{foley_o'flaherty_allen_rivera_stewart_barrangou_theriot_2021, title={Lactobacillus bile salt hydrolase substrate specificity governs bacterial fitness and host colonization}, volume={118}, ISSN={["1091-6490"]}, url={https://doi.org/10.1073/pnas.2017709118}, DOI={10.1073/pnas.2017709118}, abstractNote={Significance
          
            The transformation of bile acids (BAs) by the gut microbiota is increasingly recognized as an important factor shaping host health. The prerequisite step of BA metabolism is carried out by bile salt hydrolases (BSHs), which are encoded by select gut and probiotic bacteria. Despite their prevalence, the utility of harboring a
            bsh
            is unclear. Here, we investigate the role of BSHs encoded by
            Lactobacillus acidophilus
            and
            Lactobacillus gasseri
            . We show that BA type and BSH substrate preferences affect in vitro and in vivo growth of both species. These findings contribute to a mechanistic understanding of bacterial survival in various BA-rich niches and inform future efforts to leverage BSHs as a therapeutic tool for manipulating the gut microbiota.
          }, number={6}, journal={PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA}, publisher={Proceedings of the National Academy of Sciences}, author={Foley, Matthew H. and O'Flaherty, Sarah and Allen, Garrison and Rivera, Alissa J. and Stewart, Allison K. and Barrangou, Rodolphe and Theriot, Casey M.}, year={2021}, month={Feb} }
 @article{goh_barrangou_2021, title={Portable CRISPR-Cas9(N) System for Flexible Genome Engineering in Lactobacillus acidophilus, Lactobacillus gasseri, and Lactobacillus paracasei}, volume={87}, ISSN={["1098-5336"]}, url={https://doi.org/10.1128/AEM.02669-20}, DOI={10.1128/AEM.02669-20}, abstractNote={
            This work describes the development of a lactobacillus CRISPR-based editing system for genome manipulations in three
            Lactobacillus
            species belonging to the lactic acid bacteria (LAB), which are commonly known for their long history of use in food fermentations and as indigenous members of healthy microbiotas and for their emerging roles in human and animal commercial health-promoting applications. We exploited the established CRISPR-SpyCas9 nickase for flexible and precise genome editing applications in
            Lactobacillus acidophilus
            and further demonstrated the efficacy of this universal system in two distantly related
            Lactobacillus
            species.
          }, number={6}, journal={APPLIED AND ENVIRONMENTAL MICROBIOLOGY}, publisher={American Society for Microbiology}, author={Goh, Yong Jun and Barrangou, Rodolphe}, editor={Dudley, Edward G.Editor}, year={2021}, month={Mar} }
 @article{rubin_diamond_cress_crits-christoph_lou_borges_shivram_he_xu_zhou_et al._2021, title={Species- and site-specific genome editing in complex bacterial communities}, volume={12}, ISSN={["2058-5276"]}, DOI={10.1038/s41564-021-01014-7}, abstractNote={Understanding microbial gene functions relies on the application of experimental genetics in cultured microorganisms. However, the vast majority of bacteria and archaea remain uncultured, precluding the application of traditional genetic methods to these organisms and their interactions. Here, we characterize and validate a generalizable strategy for editing the genomes of specific organisms in microbial communities. We apply environmental transformation sequencing (ET-seq), in which nontargeted transposon insertions are mapped and quantified following delivery to a microbial community, to identify genetically tractable constituents. Next, DNA-editing all-in-one RNA-guided CRISPR–Cas transposase (DART) systems for targeted DNA insertion into organisms identified as tractable by ET-seq are used to enable organism- and locus-specific genetic manipulation in a community context. Using a combination of ET-seq and DART in soil and infant gut microbiota, we conduct species- and site-specific edits in several bacteria, measure gene fitness in a nonmodel bacterium and enrich targeted species. These tools enable editing of microbial communities for understanding and control. A suite of methods enables programmable species- and locus-specific editing of bacteria in communities.}, journal={NATURE MICROBIOLOGY}, author={Rubin, Benjamin E. and Diamond, Spencer and Cress, Brady F. and Crits-Christoph, Alexander and Lou, Yue Clare and Borges, Adair L. and Shivram, Haridha and He, Christine and Xu, Michael and Zhou, Zeyi and et al.}, year={2021}, month={Dec} }
 @article{barrangou_hill_2021, title={Todd R. Klaenhammer, an inspirational food microbiologist who leaves a lasting legacy}, volume={118}, ISSN={["0027-8424"]}, url={https://doi.org/10.1073/pnas.2107754118}, DOI={10.1073/pnas.2107754118}, abstractNote={Todd R. Klaenhammer (1951–2021) dedicated his professional life to the study of bacteria of importance to food. He conducted his doctorate under the guidance of Larry McKay at the University of Minnesota, who inspired him to apply the newest advances in bacterial genetics to the lactic acid bacteria, responsible for many food fermentations. Todd stood out from his peers at an early age. He was offered a position and started his 40-year academic career in the prestigious Food Science Department at North Carolina State University before he had even defended his doctorate. Todd always had a singular focus. While most food scientists followed the lead of the funding agencies and “better” journals by working on foodborne pathogens responsible for infectious diseases, he preferred to study the beneficial bacteria associated with food. Todd forged his illustrious career by working on commercially important bacteria, such as the dairy starter cultures responsible for cheese fermentations and probiotics that were associated with diverse health benefits. Over his long career, Todd managed to combine fundamental science with commercially relevant research. It is a measure of his accomplishments that you could spend a long time in your university library reading the many influential scientific articles Todd wrote, but if you took a lunch break and went to the dairy section of the canteen you could also choose from a variety of cheeses that were made with phage-resistant starter cultures that he generated, or perhaps enjoy a yogurt formulated with some of the health-promoting probiotics he pioneered. Throughout his career, Todd navigated the food microbiology research landscape with flair and intuition. He was always true to his favored lactic acid bacteria, but he repeatedly and skillfully adjusted the focus of his laboratory group, often leading the field into new areas. Todd was particularly attracted to using the emerging science of molecular biology to unravel the mechanisms by which these diverse bacteria play important roles in food. One of his most noteworthy pursuits included his early studies on bacteriocins that remains his most highly cited work (1). He also developed genetic engineering tools to provide the means of genetically dissecting the previously inaccessible streptococci, lactococci, and lactobacilli. These tools are still widely used in both academia and industry. Bacterial viruses (phage) were a constant source of disruption to the cheese industry, and Todd did some of his most elegant work on defining phage-resistance mechanisms and impressively managed to deploy abortive infection and restriction modification defense systems in commercial starter cultures. In this research, he even conducted some of the earliest bacterial work on RNA interference andCRISPR. He also worked extensively on the genetic basis of health-promoting lactobacilli, widely used as commercial probiotics. Much of this latter work built a foundation for the development of next-generation probiotics and Todd provided the tools used in the first series of experiments that laid the basis for the characterization of CRISPR-Cas as the bacterial adaptive immune system, fittingly in dairy cultures. Todd remained fascinated by the molecular mechanisms underpinning the interplay between bacteria and their environment, whether as hosts in need of evading predatory phages, as fermenting cultures responsible for the organoleptic properties of dairy products, or as health-promoting agents for consumers. Todd R. Klaenhammer. Image credit: North Carolina State University/Marc Hall.}, number={22}, journal={PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA}, publisher={Proceedings of the National Academy of Sciences}, author={Barrangou, Rodolphe and Hill, Colin}, year={2021}, month={Jun} }
 @article{yu_xue_barrangou_chen_huang_2021, title={Toward inclusive global governance of human genome editing}, volume={118}, ISSN={["1091-6490"]}, url={https://doi.org/10.1073/pnas.2118540118}, DOI={10.1073/pnas.2118540118}, abstractNote={In recent years, many have considered how best to govern increasingly powerful genome editing technologies. Since 2015, more than 60 statements, declarations, and other codes of practice have been published by international organizations and scientific institutions (1). In particular, the 2018 birth of two twins, Lulu and Nana—whose HIV-receptors CCR5 were altered by biophysics researcher He Jiankui—triggered widespread condemnation from the scientific community, the public, and even legal institutions. Eminent organizations that have opined on the matter include the World Health Organization’s Expert Advisory Committee on Developing Global Standards for Governance and Oversight of Human Genome Editing (WHO committee) and the International Commission on the Clinical Use of Human Germline Genome Editing (the international commission).}, number={47}, journal={PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA}, author={Yu, Hanzhi and Xue, Lan and Barrangou, Rodolphe and Chen, Shaowei and Huang, Ying}, year={2021}, month={Nov} }
 @article{brandt_barrangou_2020, title={Adaptive response to iterative passages of five Lactobacillus species in simulated vaginal fluid}, volume={20}, url={https://doi.org/10.1186/s12866-020-02027-8}, DOI={10.1186/s12866-020-02027-8}, abstractNote={AbstractBackgroundMicrobiome and metagenomic studies have given rise to a new understanding of microbial colonization of various human tissues and their ability to impact our health. One human microbiome growing in notoriety, the vaginal microbiome, stands out given its importance for women’s health, and is peculiar in terms of its relative bacterial composition, including its simplicity and typical domination by a small number ofLactobacillusspecies. The loss ofLactobacillusdominance is associated with disorders such as bacterial vaginosis, and efforts are now underway to understand the ability ofLactobacillusspecies to colonize the vaginal tract and adapt to this dynamic and acidic environment. Here, we investigate how variousLactobacillusspecies often isolated from the vaginal and intestinal cavities genomically and transcriptionally respond to iterative growth in simulated vaginal fluid.ResultsWe determined the genomes and transcriptomes ofL. acidophilus, L. crispatus, L. fermentum, L. gasseri,andL. jenseniiand compared profiles after 50, 100, 500, and 1000 generations of iterative passages in synthetic vaginal fluid. In general, we identified relatively few genetic changes consisting of single nucleotide polymorphisms, with higher counts occurring more frequently in non-vaginal isolated species. Transcriptional profiles were more impacted over time and tended to be more extensive for species that typically do not dominate the vaginal tract, reflecting a more extensive need to adapt to a less familiar environment.ConclusionsThis study provides insights into how vaginal and non-vaginalLactobacillusspecies respond and adapt to a simulated vaginal environment. Overall, trends indicate high genomic stability for all species involved, with more variability in the transcriptome especially for non-dominant species of the vaginal tract.}, number={1}, journal={BMC Microbiology}, publisher={Springer Science and Business Media LLC}, author={Brandt, Katelyn and Barrangou, Rodolphe}, year={2020}, month={Dec} }
 @article{roberts_barrangou_2020, title={Applications of CRISPR-Cas systems in lactic acid bacteria}, volume={44}, url={https://doi.org/10.1093/femsre/fuaa016}, DOI={10.1093/femsre/fuaa016}, abstractNote={ABSTRACT
               As a phenotypically and phylogenetically diverse group, lactic acid bacteria are found in a variety of natural environments and occupy important roles in medicine, biotechnology, food and agriculture. The widespread use of lactic acid bacteria across these industries fuels the need for new and functionally diverse strains that may be utilized as starter cultures or probiotics. Originally characterized in lactic acid bacteria, CRISPR-Cas systems and derived molecular machines can be used natively or exogenously to engineer new strains with enhanced functional attributes. Research on CRISPR-Cas biology and its applications has exploded over the past decade with studies spanning from the initial characterization of CRISPR-Cas immunity in Streptococcus thermophilus to the use of CRISPR-Cas for clinical gene therapies. Here, we discuss CRISPR-Cas classification, overview CRISPR biology and mechanism of action, and discuss current and future applications in lactic acid bacteria, opening new avenues for their industrial exploitation and manipulation of microbiomes.}, number={5}, journal={FEMS Microbiology Reviews}, publisher={Oxford University Press (OUP)}, author={Roberts, Avery and Barrangou, Rodolphe}, year={2020}, month={Sep}, pages={523–537} }
 @article{mcclements_barrangou_hill_kokini_lila_meyer_yu_2021, title={Building a Resilient, Sustainable, and Healthier Food Supply Through Innovation and Technology}, volume={12}, ISSN={["1941-1421"]}, DOI={10.1146/annurev-food-092220-030824}, abstractNote={The modern food supply faces many challenges. The global population continues to grow and people are becoming wealthier, so the food production system must respond by creating enough high-quality food to feed everyone with minimal damage to our environment. The number of people suffering or dying from diet-related chronic diseases, such as obesity, diabetes, heart disease, stroke, and cancer, continues to rise, which is partly linked to overconsumption of highly processed foods, especially high-calorie or rapidly digestible foods. After falling for many years, the number of people suffering from starvation or malnutrition is rising, and thishas been exacerbated by the global COVID-19 pandemic. The highly integrated food supply chains that spread around the world are susceptible to disruptions due to policy changes, economic stresses, and natural disasters, as highlighted by the recent pandemic. In this perspective article, written by members of the Editorial Committee of the Annual Review of Food Science and Technology, we highlight some of the major challenges confronting the modern food supply chain as well as how innovations in policy and technology can be used to address them. Pertinent technological innovations include robotics, machine learning, artificial intelligence, advanced diagnostics, nanotechnology, biotechnology, gene editing, vertical farming, and soft matter physics. Many of these technologies are already being employed across the food chain by farmers, distributors, manufacturers, and consumers to improve the quality, nutrition, safety, and sustainability of the food supply. These innovations are required to stimulate the development and implementation of new technologies to ensure a more equitable, resilient, and efficient food production system. Where appropriate, these technologies should be carefully tested before widespread implementation so that proper risk–benefit analyses can be carried out. They can then be employed without causing unforeseen adverse consequences. Finally, it is important to actively engage all stakeholders involved in the food supply chain throughout the development and testing of these new technologies to support their adoption if proven safe and effective.}, journal={ANNUAL REVIEW OF FOOD SCIENCE AND TECHNOLOGY, VOL 12, 2021}, author={McClements, David Julian and Barrangou, Rodolphe and Hill, Colin and Kokini, Jozef L. and Lila, Mary Ann and Meyer, Anne S. and Yu, Liangli}, year={2021}, pages={1–28} }
 @article{barrangou_sontheimer_2020, title={CRISPR Shields: Fending Off Diverse Cas Nucleases with Nucleus-like Structures}, volume={77}, ISSN={["1097-4164"]}, DOI={10.1016/j.molcel.2020.02.015}, abstractNote={Two recent studies have uncovered a novel means by which bacteriophages thwart host immunity. Mendoza et al., 2020Mendoza S.D. Nieweglowska E.S. Govindarajan S. Leon L.M. Berry J.D. Tiwari A. Chaikeeratisak V. Pogliano J. Agard D.A. Bondy-Denomy J. A bacteriophage nucleus-like compartment shields DNA from CRISPR nucleases.Nature. 2020; 577: 244-248Crossref PubMed Scopus (71) Google Scholar and Malone et al., 2020Malone L.M. Warring S.L. Jackson S.A. Warnecke C. Gardner P.P. Gumy L.F. Fineran P.C. A jumbo phage that forms a nucleus-like structure evades CRISPR-Cas DNA targeting but is vulnerable to type III RNA-based immunity.Nat. Microbiol. 2020; 5: 48-55Crossref PubMed Scopus (69) Google Scholar demonstrate that a nucleus-like proteinaceous structure shields phage DNA from CRISPR-associated nucleases encompassing Cascade-Cas3, Cas9, and Cas12. Two recent studies have uncovered a novel means by which bacteriophages thwart host immunity. Mendoza et al., 2020Mendoza S.D. Nieweglowska E.S. Govindarajan S. Leon L.M. Berry J.D. Tiwari A. Chaikeeratisak V. Pogliano J. Agard D.A. Bondy-Denomy J. A bacteriophage nucleus-like compartment shields DNA from CRISPR nucleases.Nature. 2020; 577: 244-248Crossref PubMed Scopus (71) Google Scholar and Malone et al., 2020Malone L.M. Warring S.L. Jackson S.A. Warnecke C. Gardner P.P. Gumy L.F. Fineran P.C. A jumbo phage that forms a nucleus-like structure evades CRISPR-Cas DNA targeting but is vulnerable to type III RNA-based immunity.Nat. Microbiol. 2020; 5: 48-55Crossref PubMed Scopus (69) Google Scholar demonstrate that a nucleus-like proteinaceous structure shields phage DNA from CRISPR-associated nucleases encompassing Cascade-Cas3, Cas9, and Cas12. The dynamic arms race between prokaryotic hosts and viruses is well chronicled (Hampton et al., 2020Hampton H.G. Watson B.N.J. Fineran P.C. The arms race between bacteria and their phage foes.Nature. 2020; 577: 327-336Crossref PubMed Scopus (240) Google Scholar), especially with the recent addition of CRISPR-Cas adaptive immune systems (Makarova et al., 2020Makarova K.S. Wolf Y.I. Iranzo J. Shmakov S.A. Alkhnbashi O.S. Brouns S.J.J. Charpentier E. Cheng D. Haft D.H. Horvath P. et al.Evolutionary classification of CRISPR-Cas systems: a burst of class 2 and derived variants..Nat. Rev. Microbiol. 2020; 18: 435-448Crossref Scopus (733) Google Scholar) and the subsequent discovery that bacteriophages deploy countermeasures enabling CRISPR escape or evasion (Hampton et al., 2020Hampton H.G. Watson B.N.J. Fineran P.C. The arms race between bacteria and their phage foes.Nature. 2020; 577: 327-336Crossref PubMed Scopus (240) Google Scholar, Stanley and Maxwell, 2018Stanley S.Y. Maxwell K.L. Phage-Encoded Anti-CRISPR Defenses.Annu. Rev. Genet. 2018; 52: 445-464Crossref PubMed Scopus (83) Google Scholar). The broad and diverse CRISPR-Cas arsenal featured in many bacteria and most archaea reflects the evolutionary value of this adaptive immune system, especially in protecting against widespread lytic bacteriophages. To cope with the range of CRISPR defense systems, several studies have revealed a plethora of bacteriophage escape mechanisms, encompassing anti-CRISPR inhibitors, recombination between genetically distinct genomes, and specific mutations at Cas nuclease binding and targeting sites. Now, expanding the CRISPR evasion menu, two studies (Mendoza et al., 2020Mendoza S.D. Nieweglowska E.S. Govindarajan S. Leon L.M. Berry J.D. Tiwari A. Chaikeeratisak V. Pogliano J. Agard D.A. Bondy-Denomy J. A bacteriophage nucleus-like compartment shields DNA from CRISPR nucleases.Nature. 2020; 577: 244-248Crossref PubMed Scopus (71) Google Scholar, Malone et al., 2020Malone L.M. Warring S.L. Jackson S.A. Warnecke C. Gardner P.P. Gumy L.F. Fineran P.C. A jumbo phage that forms a nucleus-like structure evades CRISPR-Cas DNA targeting but is vulnerable to type III RNA-based immunity.Nat. Microbiol. 2020; 5: 48-55Crossref PubMed Scopus (69) Google Scholar) show that some phages with unusually large genomes, "jumbophages," can form nucleus-like shells that physically shield viral DNA from CRISPR-Cas effectors, as well as from restriction endonucleases that also defend against phage predation (Figure 1). Furthermore, the subset of CRISPR-Cas systems that degrade RNA (in addition to, or instead of, phage DNA) retain their efficacy in the face of these nucleus-like jumbophage shells, partially explaining the evolutionary pressures driving the emergence and spread of RNA-targeting CRISPR-Cas machineries. Bondy-Denomy et al., 2013Bondy-Denomy J. Pawluk A. Maxwell K.L. Davidson A.R. Bacteriophage genes that inactivate the CRISPR/Cas bacterial immune system.Nature. 2013; 493: 429-432Crossref PubMed Scopus (501) Google Scholar had previously used Pseudomonas aeruginosa and its phages to discover anti-CRISPR proteins, which are now known to be widespread (Stanley and Maxwell, 2018Stanley S.Y. Maxwell K.L. Phage-Encoded Anti-CRISPR Defenses.Annu. Rev. Genet. 2018; 52: 445-464Crossref PubMed Scopus (83) Google Scholar). Most anti-CRISPRs bind CRISPR-Cas effectors and prevent target nucleic acid recognition or degradation. Working again in P. aeruginosa, Bondy-Denomy and colleagues began their more recent study (Mendoza et al., 2020Mendoza S.D. Nieweglowska E.S. Govindarajan S. Leon L.M. Berry J.D. Tiwari A. Chaikeeratisak V. Pogliano J. Agard D.A. Bondy-Denomy J. A bacteriophage nucleus-like compartment shields DNA from CRISPR nucleases.Nature. 2020; 577: 244-248Crossref PubMed Scopus (71) Google Scholar) with a successful search for phages that can circumvent the immunity conferred by type I-C CRISPR-Cas systems that are native to some strains of this opportunistic pathogen. A jumbophage called φKZ successfully counteracted type I-C CRISPR-Cas immunity, making it a potential source of anti-CRISPRs specific for this subtype. Surprisingly, however, infections of control strains revealed φKZ to be similarly impervious to endogenous type I-F CRISPR-Cas defenses, as well as heterologous type II-A and type V-A CRISPR-Cas machineries and even restriction enzymes. The range of nucleases encompassed here spans the CRISPR-Cas effectors Cascade-Cas3, Cas9, and Cas12, as well as "classical" type I and type II restriction enzymes (EcoRI, HsdRMS), illustrating how widely effective this "pan-resistance" mechanism may be. More importantly, the unprecedented mechanistic and phylogenetic breadth of inhibition would be difficult to explain for a conventional anti-CRISPR protein, leading Mendoza et al. to seek other explanations for such impressive resilience against diverse host immunity. Intriguingly, certain jumbophages that infect P. aeruginosa had been shown previously to form an intracellular, proteinaceous shell that surrounds the phage genome during lytic growth and DNA replication (Chaikeeratisak et al., 2017Chaikeeratisak V. Nguyen K. Khanna K. Brilot A.F. Erb M.L. Coker J.K. Vavilina A. Newton G.L. Buschauer R. Pogliano K. et al.Assembly of a nucleus-like structure during viral replication in bacteria.Science. 2017; 355: 194-197Crossref PubMed Scopus (110) Google Scholar). Mechanistically, this nucleus-like structure is a multimeric assembly of a single protein unit and is localized to the cell center by PhuZ, a phage-encoded tubulin homolog. This physically defined shell-like structural feature has the potential to protect phage φKZ DNA by providing a shield preventing exposure to nucleases. This structure houses only the phage (and not the host) DNA, and the barrier mechanism is somewhat selective, allowing entry of important enzymes critical for viral DNA replication and progeny particle assembly, yet excluding many others (Chaikeeratisak et al., 2017Chaikeeratisak V. Nguyen K. Khanna K. Brilot A.F. Erb M.L. Coker J.K. Vavilina A. Newton G.L. Buschauer R. Pogliano K. et al.Assembly of a nucleus-like structure during viral replication in bacteria.Science. 2017; 355: 194-197Crossref PubMed Scopus (110) Google Scholar). Mendoza et al. demonstrated that fluorescently tagged CRISPR-Cas effectors and restriction endonucleases are likewise excluded from the shell's interior, suggesting physical sequestration of phage DNA as the broadly effective anti-immunity mechanism. Furthermore, extracted phage DNA was found to be susceptible to Cas9 and restriction endonuclease cleavage in vitro, demonstrating that shell-encased DNA is not intrinsically resistant to enzymatic digestion, e.g., via chemical modification. To establish a causal role for genome sequestration as an anti-immunity countermeasure, the authors sent a restriction enzyme (EcoRI) "under the radar" by fusing it to a phage recombinase (ORF152, encoding a RecA homolog) shown previously to be shell-permeable. Whereas EcoRI alone was excluded from the shell and ineffective in host defense, the EcoRI-ORF152 chimera trafficked into the nucleus-like structure and conferred substantial host immunity. The finding that intra-shell transport of a DNA-targeting immune effector negates the φKZ countermeasures clearly implicates physical DNA sequestration as the jumbophage's anti-immunity mechanism. Nonetheless, the ORF152 fusion approach proved unsuccessful when applied to the larger Cas9 nuclease, suggesting size limits to active shell permeability. Similarly, Fineran and colleagues (Malone et al., 2020Malone L.M. Warring S.L. Jackson S.A. Warnecke C. Gardner P.P. Gumy L.F. Fineran P.C. A jumbo phage that forms a nucleus-like structure evades CRISPR-Cas DNA targeting but is vulnerable to type III RNA-based immunity.Nat. Microbiol. 2020; 5: 48-55Crossref PubMed Scopus (69) Google Scholar) explored phage resistance to CRISPR-Cas immunity in a strain from the genus Serratia and identified a jumbophage that encodes a tubulin homolog, forms a microscopically visible shell, and escapes CRISPR targeting by the Cascade-Cas3 effectors of endogenous type I-E and I-F systems. These results further implicate nucleus-like shells in negating CRISPR-Cas immunity and extend this strategy beyond P. aeruginosa jumbophages. Future studies will be needed to determine how this physical structure is assembled, how its barrier function is gated, and how widespread this mechanism is. Mechanistic explorations of CRISPR-Cas immunity have revealed some systems (type III) that augment the DNA-cleaving activity of their Cas10 effectors with separate ribonuclease activities and others (type VI) that rely solely on RNA cleavage by Cas13, without benefit of DNA degradation activity (Makarova et al., 2020Makarova K.S. Wolf Y.I. Iranzo J. Shmakov S.A. Alkhnbashi O.S. Brouns S.J.J. Charpentier E. Cheng D. Haft D.H. Horvath P. et al.Evolutionary classification of CRISPR-Cas systems: a burst of class 2 and derived variants..Nat. Rev. Microbiol. 2020; 18: 435-448Crossref Scopus (733) Google Scholar). Intriguingly, although jumbophage DNA replication and transcription are thought to occur inside the shell, phage-derived mRNAs must escape the shell for translation, as ribosomes do not appear to be imported (Chaikeeratisak et al., 2017Chaikeeratisak V. Nguyen K. Khanna K. Brilot A.F. Erb M.L. Coker J.K. Vavilina A. Newton G.L. Buschauer R. Pogliano K. et al.Assembly of a nucleus-like structure during viral replication in bacteria.Science. 2017; 355: 194-197Crossref PubMed Scopus (110) Google Scholar). Might this imply that RNA-targeting systems provide hosts with immune systems that nucleus-like shells cannot circumvent? Using a heterologous Cas13 effector and an endogenous Cas10-based system, respectively, Mendoza et al. and Malone et al. demonstrate that the answer to this question is yes: in both cases, CRISPR-Cas immunity based on RNA degradation persists during jumbophage infection. This observation provides a compelling rationale for the evolution of RNA-targeting CRISPR-Cas systems, alongside their capacity to induce altruistic dormancy responses via host transcript targeting (Meeske et al., 2019Meeske A.J. Nakandakari-Higa S. Marraffini L.A. Cas13-induced cellular dormancy prevents the rise of CRISPR-resistant bacteriophage.Nature. 2019; 570: 241-245Crossref PubMed Scopus (112) Google Scholar, Rostøl and Marraffini, 2019Rostøl J.T. Marraffini L.A. Non-specific degradation of transcripts promotes plasmid clearance during type III-A CRISPR-Cas immunity.Nat. Microbiol. 2019; 4: 656-662Crossref PubMed Scopus (76) Google Scholar). These studies further illustrate how the interplay between bacteria and predatory bacteriophages continues to provide insights into the dynamic arms race playing out globally between various microbiomes and viromes that shape the composition and biology of most habitats on the planet. Furthermore, they open new avenues for the understanding of CRISPR-Cas systems and their impact on phage-host dynamics, and also illustrate how much potential lies in the continued exploration of new hosts, wild phages, and recently discovered defense systems (Doron et al., 2018Doron S. Melamed S. Ofir G. Leavitt A. Lopatina A. Keren M. Amitai G. Sorek R. Systematic discovery of antiphage defense systems in the microbial pangenome.Science. 2018; 359: eaar4120Crossref PubMed Scopus (393) Google Scholar). R.B. acknowledges support from NC State University, and E.J.S. acknowledges funding from the NIH (GM125797). The authors also acknowledge graphical support from Avery Roberts in the CRISPR lab. R.B. is a shareholder of Caribou Biosciences, Intellia Therapeutics, Locus Biosciences, Inari Ag, and TreeCo. E.J.S. is a shareholder of Intellia Therapeutics.}, number={5}, journal={MOLECULAR CELL}, author={Barrangou, Rodolphe and Sontheimer, Erik J.}, year={2020}, month={Mar}, pages={934–936} }
 @article{hidalgo-cantabrana_barrangou_2020, title={Characterization and applications of Type I CRISPR-Cas systems}, volume={48}, url={https://doi.org/10.1042/BST20190119}, DOI={10.1042/BST20190119}, abstractNote={CRISPR-Cas constitutes the adaptive immune system of bacteria and archaea. This RNA-mediated sequence-specific recognition and targeting machinery has been used broadly for diverse applications in a wide range of organisms across the tree of life. The compact class 2 systems, that hinge on a single Cas effector nuclease have been harnessed for genome editing, transcriptional regulation, detection, imaging and other applications, in different research areas. However, most of the CRISPR-Cas systems belong to class 1, and the molecular machinery of the most widespread and diverse Type I systems afford tremendous opportunities for a broad range of applications. These highly abundant systems rely on a multi-protein effector complex, the CRISPR associated complex for antiviral defense (Cascade), which drives DNA targeting and cleavage. The complexity of these systems has somewhat hindered their widespread usage, but the pool of thousands of diverse Type I CRISPR-Cas systems opens new avenues for CRISPR-based applications in bacteria, archaea and eukaryotes. Here, we describe the features and mechanism of action of Type I CRISPR-Cas systems, illustrate how endogenous systems can be reprogrammed to target the host genome and perform genome editing and transcriptional regulation by co-delivering a minimal CRISPR array together with a repair template. Moreover, we discuss how these systems can also be used in eukaryotes. This review provides a framework for expanding the CRISPR toolbox, and repurposing the most abundant CRISPR-Cas systems for a wide range of applications.}, number={1}, journal={Biochemical Society Transactions}, publisher={Portland Press Ltd.}, author={Hidalgo-Cantabrana, Claudio and Barrangou, Rodolphe}, year={2020}, month={Feb}, pages={15–23} }
 @misc{pan_barrangou_2020, title={Combining omics technologies with CRISPR-based genome editing to study food microbes}, volume={61}, ISSN={["1879-0429"]}, DOI={10.1016/j.copbio.2019.12.027}, abstractNote={The implementation of omics technologies such as genomics, proteomics and transcriptomics has revolutionized our understanding of microbiomes, and shed light on the functional attributes and mechanisms of action underlying the ability of probiotics to impact host health and starter cultures to drive food fermentation. Recently, molecular machines from CRISPR-Cas systems have redefined the gene editing toolbox and democritized our ability to alter the genome of food microorganisms. An integrated approach in which CRISPR-based genome editing is informed by omics studies is poised to enable the engineering of microorganisms and the formulation of microbiomes impacting the food supply chain. Here, we highlight the current applications of omics technologies in food microorganisms and CRISPR-based genome editing technologies in bacteria, and discuss how this integrated approach enables effective engineering of food microbes to generate enhanced probiotic strains, develop novel biotherapeutics and alter microbial communities in food matrices.}, journal={CURRENT OPINION IN BIOTECHNOLOGY}, author={Pan, Meichen and Barrangou, Rodolphe}, year={2020}, month={Feb}, pages={198–208} }
 @article{pan_hidalgo-cantabrana_goh_sanozky-dawes_barrangou_2020, title={Comparative Analysis of Lactobacillus gasseri and Lactobacillus crispatus Isolated From Human Urogenital and Gastrointestinal Tracts}, volume={10}, ISSN={["1664-302X"]}, DOI={10.3389/fmicb.2019.03146}, abstractNote={Lactobacillus crispatus and Lactobacillus gasseri are two of the main Lactobacillus species found in the healthy vaginal microbiome and have also previously been identified and isolated from the human gastrointestinal (GI) tract. These two ecological niches are fundamentally different, notably with regards to the epithelial cell type, nutrient availability, environmental conditions, pH, and microbiome composition. Given the dramatic differences between these two environments, we characterized strains within the same Lactobacillus species isolated from either the vaginal or intestinal tract to assess whether they are phenotypically and genetically different. We compared the genomes of the Lactobacillus strains selected in this study for genetic features of interest, and performed a series of comparative phenotypic assays including small intestinal juice and acid resistance, carbohydrate fermentation profiles, lactic acid production, and host interaction with intestinal Caco-2 and vaginal VK2 cell lines. We also developed a simulated vaginal fluid (SVF) to study bacterial growth in a proxy vaginal environment and conducted differential transcriptomic analysis between SVF and standard laboratory MRS medium. Overall, our results show that although strain-specific variation is observed, some phenotypic differences seem associated with the isolation source. We encourage future probiotic formulation to include isolation source and take into consideration genetic and phenotypic features for use at various body sites.}, journal={FRONTIERS IN MICROBIOLOGY}, author={Pan, Meichen and Hidalgo-Cantabrana, Claudio and Goh, Yong Jun and Sanozky-Dawes, Rosemary and Barrangou, Rodolphe}, year={2020}, month={Jan} }
 @article{o'flaherty_foley_rivera_theriot_barrangou_2020, title={Complete Genome Sequence of Lactobacillus johnsonii NCK2677, Isolated from Mice}, volume={9}, url={https://doi.org/10.1128/MRA.00918-20}, DOI={10.1128/MRA.00918-20}, abstractNote={
            We report the closed genome sequence of a
            Lactobacillus johnsonii
            strain (NCK2677) that was isolated from a cefoperazone-treated mouse model designed for the study of
            Clostridioides difficile
            infection. Illumina and Nanopore sequencing reads were assembled into a circular 1,951,416-bp chromosome with a G+C content of 34.7%, containing 1,865 genes.
          }, number={43}, journal={Microbiology Resource Announcements}, publisher={American Society for Microbiology}, author={O'Flaherty, Sarah and Foley, Matthew H. and Rivera, Alissa J. and Theriot, Casey M. and Barrangou, Rodolphe}, editor={Rasko, DavidEditor}, year={2020}, month={Oct} }
 @article{pan_nethery_hidalgo-cantabrana_barrangou_2020, title={Comprehensive Mining and Characterization of CRISPR-Cas Systems in Bifidobacterium}, volume={8}, url={https://doi.org/10.3390/microorganisms8050720}, DOI={10.3390/microorganisms8050720}, abstractNote={The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas (CRISPR-associated cas) systems constitute the adaptive immune system in prokaryotes, which provides resistance against bacteriophages and invasive genetic elements. The landscape of applications in bacteria and eukaryotes relies on a few Cas effector proteins that have been characterized in detail. However, there is a lack of comprehensive studies on naturally occurring CRISPR-Cas systems in beneficial bacteria, such as human gut commensal Bifidobacterium species. In this study, we mined 954 publicly available Bifidobacterium genomes and identified CRIPSR-Cas systems in 57% of these strains. A total of five CRISPR-Cas subtypes were identified as follows: Type I-E, I-C, I-G, II-A, and II-C. Among the subtypes, Type I-C was the most abundant (23%). We further characterized the CRISPR RNA (crRNA), tracrRNA, and PAM sequences to provide a molecular basis for the development of new genome editing tools for a variety of applications. Moreover, we investigated the evolutionary history of certain Bifidobacterium strains through visualization of acquired spacer sequences and demonstrated how these hypervariable CRISPR regions can be used as genotyping markers. This extensive characterization will enable the repurposing of endogenous CRISPR-Cas systems in Bifidobacteria for genome engineering, transcriptional regulation, genotyping, and screening of rare variants.}, number={5}, journal={Microorganisms}, publisher={MDPI AG}, author={Pan, Meichen and Nethery, Matthew A. and Hidalgo-Cantabrana, Claudio and Barrangou, Rodolphe}, year={2020}, month={May}, pages={720} }
 @article{klotz_goh_o'flaherty_johnson_barrangou_2020, title={Deletion of S-Layer Associated Ig-Like Domain Protein Disrupts the Lactobacillus acidophilus Cell Surface}, volume={11}, ISSN={["1664-302X"]}, DOI={10.3389/fmicb.2020.00345}, abstractNote={Bacterial surface-layers (S-layers) are crystalline arrays of repeating proteinaceous subunits that coat the exterior of many cell envelopes. S-layers have demonstrated diverse functions in growth and survival, maintenance of cell integrity, and mediation of host interactions. Additionally, S-layers can act as scaffolds for the outward display of auxiliary proteins and glycoproteins. These non-covalently bound S-layer associated proteins (SLAPs) have characterized roles in cell division, adherence to intestinal cells, and modulation of the host immune response. Recently, IgdA (LBA0695), a Lactobacillus acidophilus SLAP that possesses a Group 3 immunoglobulin (Ig)-like domain and GW (Gly-Tryp) dipeptide surface anchor, was recognized for its high conservation among S-layer-forming lactobacilli, constitutive expression, and surface localization. These findings prompted its selection for examination within the present study. Although IgdA and corresponding orthologs were shown to be unique to host-adapted lactobacilli, the Ig domain itself was specific to vertebrate-adapted species suggesting a role in vertebrate adaptation. Using a counterselective gene replacement system, igdA was deleted from the L. acidophilus NCFM chromosome. The resultant mutant, NCK2532, exhibited a visibly disrupted cell surface which likely contributed to its higher salt sensitivity, severely reduced adhesive capacity, and altered immunogenicity profile. Transcriptomic analyses revealed the induction of several stress response genes and secondary surface proteins. Due to the broad impact of IgdA on the cellular physiology and probiotic attributes of L. acidophilus, identification of similar proteins in alternative bacterial species may help pinpoint next-generation host-adapted probiotic candidates.}, journal={FRONTIERS IN MICROBIOLOGY}, author={Klotz, Courtney and Goh, Yong Jun and O'Flaherty, Sarah and Johnson, Brant and Barrangou, Rodolphe}, year={2020}, month={Mar} }
 @article{brandt_nethery_sarah_barrangou_2020, title={Genomic characterization of Lactobacillus fermentum DSM 20052}, volume={21}, url={https://doi.org/10.1186/s12864-020-6740-8}, DOI={10.1186/s12864-020-6740-8}, abstractNote={Abstract
Background
Lactobacillus fermentum, a member of the lactic acid bacteria complex, has recently garnered increased attention due to documented antagonistic properties and interest in assessing the probiotic potential of select strains that may provide human health benefits. Here, we genomically characterize L. fermentum using the type strain DSM 20052 as a canonical representative of this species.

Results
We determined the polished whole genome sequence of this type strain and compared it to 37 available genome sequences within this species. Results reveal genetic diversity across nine clades, with variable content encompassing mobile genetic elements, CRISPR-Cas immune systems and genomic islands, as well as numerous genome rearrangements. Interestingly, we determined a high frequency of occurrence of diverse Type I, II, and III CRISPR-Cas systems in 72% of the genomes, with a high level of strain hypervariability.

Conclusions
These findings provide a basis for the genetic characterization of L. fermentum strains of scientific and commercial interest. Furthermore, our study enables genomic-informed selection of strains with specific traits for commercial product formulation, and establishes a framework for the functional characterization of features of interest.
}, number={1}, journal={BMC Genomics}, publisher={Springer Science and Business Media LLC}, author={Brandt, Katelyn and Nethery, Matthew A. and Sarah, O’Flaherty and Barrangou, Rodolphe}, year={2020}, month={Dec} }
 @article{pan_hidalgo-cantabrana_barrangou_2020, title={Host and body site-specific adaptation of Lactobacillus crispatus genomes}, volume={2}, url={https://doi.org/10.1093/nargab/lqaa001}, DOI={10.1093/nargab/lqaa001}, abstractNote={Abstract
               Lactobacillus crispatus is a common inhabitant of both healthy poultry gut and human vaginal tract, and the absence of this species has been associated with a higher risk of developing infectious diseases. In this study, we analyzed 105 L. crispatus genomes isolated from a variety of ecological niches, including the human vaginal tract, human gut, chicken gut and turkey gut, to shed light on the genetic and functional features that drive evolution and adaptation of this important species. We performed in silico analyses to identify the pan and core genomes of L. crispatus, and to reveal the genomic differences and similarities associated with their origins of isolation. Our results demonstrated that, although a significant portion of the genomic content is conserved, human and poultry L. crispatus isolates evolved to encompass different genomic features (e.g. carbohydrate usage, CRISPR–Cas immune systems, prophage occurrence) in order to thrive in different environmental niches. We also observed that chicken and turkey L. crispatus isolates can be differentiated based on their genomic information, suggesting significant differences may exist between these two poultry gut niches. These results provide insights into host and niche-specific adaptation patterns in species of human and animal importance.}, number={1}, journal={NAR Genomics and Bioinformatics}, publisher={Oxford University Press (OUP)}, author={Pan, Meichen and Hidalgo-Cantabrana, Claudio and Barrangou, Rodolphe}, year={2020}, month={Mar} }
 @article{selle_fletcher_tuson_schmitt_mcmillan_vridhambal_rivera_montgomery_fortier_barrangou_et al._2020, title={In Vivo Targeting of Clostridioides difficile Using Phage-Delivered CRISPR-Cas3 Antimicrobials}, volume={11}, url={https://doi.org/10.1128/mBio.00019-20}, DOI={10.1128/mBio.00019-20}, abstractNote={
            Clostridioides difficile
            is a bacterial pathogen responsible for significant morbidity and mortality across the globe. Current therapies based on broad-spectrum antibiotics have some clinical success, but approximately 30% of patients have relapses, presumably due to the continued perturbation to the gut microbiota. Here, we show that phages can be engineered with type I CRISPR-Cas systems and modified to reduce lysogeny and to enable the specific and efficient targeting and killing of
            C. difficile
            in vitro
            and
            in vivo.
            Additional genetic engineering to disrupt phage modulation of toxin expression by lysogeny or other mechanisms would be required to advance a CRISPR-enhanced phage antimicrobial for
            C. difficile
            toward clinical application. These findings provide evidence into how phage can be combined with CRISPR-based targeting to develop novel therapies and modulate microbiomes associated with health and disease.
          }, number={2}, journal={mBio}, publisher={American Society for Microbiology}, author={Selle, Kurt and Fletcher, Joshua R. and Tuson, Hannah and Schmitt, Daniel S. and McMillan, Lana and Vridhambal, Gowrinarayani S. and Rivera, Alissa J. and Montgomery, Stephanie A. and Fortier, Louis-Charles and Barrangou, Rodolphe and et al.}, editor={Ballard, Jimmy D.Editor}, year={2020}, month={Apr} }
 @article{angrist_barrangou_baylis_brokowski_burgio_caplan_chapman_church_cook-deegan_cwik_et al._2020, title={Reactions to the National Academies/Royal Society Report on Heritable Human Genome Editing}, volume={3}, ISSN={["2573-1602"]}, DOI={10.1089/crispr.2020.29106.man}, abstractNote={In September 2020, a detailed report on Heritable Human Genome Editing was published. The report offers a translational pathway for the limited approval of germline editing under limited circumstances and assuming various criteria have been met. In this perspective, some three dozen experts from the fields of genome editing, medicine, bioethics, law, and related fields offer their candid reactions to the National Academies/Royal Society report, highlighting areas of support, omissions, disagreements, and priorities moving forward.}, number={5}, journal={CRISPR JOURNAL}, author={Angrist, Misha and Barrangou, Rodolphe and Baylis, Francoise and Brokowski, Carolyn and Burgio, Gaetan and Caplan, Arthur and Chapman, Carolyn Riley and Church, George M. and Cook-Deegan, Robert and Cwik, Bryan and et al.}, year={2020}, month={Oct}, pages={332–349} }
 @article{klotz_goh_sarah_barrangou_2020, title={S-layer associated proteins contribute to the adhesive and immunomodulatory properties of Lactobacillus acidophilus NCFM}, volume={20}, url={https://doi.org/10.1186/s12866-020-01908-2}, DOI={10.1186/s12866-020-01908-2}, abstractNote={AbstractBackgroundSurface layers (S-layers) are two-dimensional crystalline arrays of repeating proteinaceous subunits that form the outermost layer of many bacterial cell envelopes. Within theLactobacillusgenus, S-layer presence is frequently associated with probiotic-relevant properties such as improved adherence to host epithelial cells and modulation of the immune response. However, recent studies have demonstrated that certain S-layer functions may be supplemented by a novel subset of proteins embedded within its lattice, termed S-layer associated proteins (SLAPs). In the following study, fourLactobacillus acidophilusNCFM SLAPs (LBA0046, LBA0864, LBA1426, and LBA1539) were selected for in silico and phenotypic assessment.ResultsDespite lacking any sequence similarity or catalytic domains that may indicate function, the genes encoding the four proteins of interest were shown to be unique to S-layer-forming, host-adapted lactobacilli species. Likewise, their corresponding deletion mutants exhibited broad, host-relevant phenotypes including decreased inflammatory profiles and reduced adherence to Caco-2 intestinal cells, extracellular matrices, and mucin in vitro.ConclusionsOverall, the data presented in this study collectively links several previously uncharacterized extracellular proteins to roles in the underlying host adaptive mechanisms ofL. acidophilus.}, number={1}, journal={BMC Microbiology}, publisher={Springer Science and Business Media LLC}, author={Klotz, Courtney and Goh, Yong Jun and Sarah, O’Flaherty and Barrangou, Rodolphe}, year={2020}, month={Dec} }
 @article{lamanna_pyhtila_barrangou_2020, title={Sharing the CRISPR Toolbox with an Expanding Community}, volume={3}, ISSN={["2573-1602"]}, DOI={10.1089/crispr.2020.0075}, abstractNote={Over the past 8 years, the widespread adoption of CRISPR-based technologies has fueled the global genome editing revolution. This platform is based on Cas molecular machines such as Cas9, Cas12, Cas13, as well as other CRISPR effector proteins that are able to alter the genome, transcriptome, and epigenome of virtually any species. Technological improvements have rendered these tools more efficient and precise, and enabled functional diversification and specialization, as recently illustrated by the rise of base editing and the quickly growing demand for prime editing constructs. Here, we discuss the continued adoption of CRISPR tools and constructs distributed by the nonprofit organization Addgene, highlight the trends in the global demand for the CRISPR toolbox, and consider the widespread attitude changes around open sharing that are having a transformative effect on speeding up science.}, number={4}, journal={CRISPR JOURNAL}, author={LaManna, Caroline M. and Pyhtila, Brook and Barrangou, Rodolphe}, year={2020}, month={Aug}, pages={248–252} }
 @article{barrangou_sontheimer_2020, title={Shutting down RNA-targeting CRISPR}, volume={369}, ISSN={["1095-9203"]}, DOI={10.1126/science.abc8243}, abstractNote={The discovery of an anti-CRISPR reveals viral escape from CRISPR immunity}, number={6499}, journal={SCIENCE}, author={Barrangou, Rodolphe and Sontheimer, Erik J.}, year={2020}, month={Jul}, pages={31–32} }
 @article{reed_nethery_stewart_barrangou_theriot_2020, title={Strain-Dependent Inhibition of Clostridioides difficile by Commensal Clostridia Carrying the Bile Acid-Inducible ( bai ) Operon}, volume={202}, url={https://doi.org/10.1128/JB.00039-20}, DOI={10.1128/JB.00039-20}, abstractNote={ABSTRACTClostridioides difficileis one of the leading causes of antibiotic-associated diarrhea. Gut microbiota-derived secondary bile acids and commensalClostridiathat carry the bile acid-inducible (bai) operon are associated with protection fromC. difficileinfection (CDI), although the mechanism is not known. In this study, we hypothesized that commensalClostridiaare important for providing colonization resistance againstC. difficiledue to their ability to produce secondary bile acids, as well as potentially competing againstC. difficilefor similar nutrients. To test this hypothesis, we examined the abilities of four commensalClostridiacarrying thebaioperon (Clostridium scindensVPI 12708,C. scindensATCC 35704,Clostridium hiranonis, andClostridium hylemonae) to convert cholate (CA) to deoxycholate (DCA)in vitro,and we determined whether the amount of DCA produced was sufficient to inhibit the growth of a clinically relevantC. difficilestrain. We also investigated the competitive relationships between these commensals andC. difficileusing anin vitrococulture system. We found that inhibition ofC. difficilegrowth by commensalClostridiasupplemented with CA was strain dependent, correlated with the production of ∼2 mM DCA, and increased the expression ofbaioperon genes. We also found thatC. difficilewas able to outcompete all four commensalClostridiain anin vitrococulture system. These studies are instrumental in understanding the relationship between commensalClostridiaandC. difficilein the gut, which is vital for designing targeted bacterial therapeutics. Future studies dissecting the regulation of thebaioperonin vitroandin vivoand how this affects CDI will be important.IMPORTANCECommensalClostridiacarrying thebaioperon, such asC. scindens,have been associated with protection against CDI; however, the mechanism for this protection is unknown. Herein, we show four commensalClostridiathat carry thebaioperon and affectC. difficilegrowth in a strain-dependent manner, with and without the addition of cholate. Inhibition ofC. difficileby commensals correlated with the efficient conversion of cholate to deoxycholate, a secondary bile acid that inhibitsC. difficilegermination, growth, and toxin production. Competition studies also revealed thatC. difficilewas able to outcompete the commensals in anin vitrococulture system. These studies are instrumental in understanding the relationship between commensalClostridiaandC. difficilein the gut, which is vital for designing targeted bacterial therapeutics.}, number={11}, journal={Journal of Bacteriology}, publisher={American Society for Microbiology}, author={Reed, A. D. and Nethery, M. A. and Stewart, A. and Barrangou, R. and Theriot, C. M.}, editor={Comstock, Laurie E.Editor}, year={2020}, month={May} }
 @article{reed_nethery_stewart_barrangou_theriot_2020, title={Strain-dependent inhibition ofClostridioides difficileby commensalClostridiaencoding the bile acid inducible(bai)operon}, url={https://doi.org/10.1101/2020.01.22.916304}, DOI={10.1101/2020.01.22.916304}, abstractNote={AbstractClostridioides difficile is one of the leading causes of antibiotic-associated diarrhea. Gut microbiota-derived secondary bile acids and commensal Clostridia that encode the bile acid inducible (bai) operon are associated with protection from C. difficile infection (CDI), although the mechanism is not known. In this study we hypothesized that commensal Clostridia are important for providing colonization resistance against C. difficile due to their ability to produce secondary bile acids, as well as potentially competing against C. difficile for similar nutrients. To test this hypothesis, we examined the ability of four commensal Clostridia encoding the bai operon (C. scindens VPI 12708, C. scindens ATCC 35704, C. hiranonis, and C. hylemonae) to convert CA to DCA in vitro, and if the amount of DCA produced was sufficient to inhibit growth of a clinically relevant C. difficile strain. We also investigated the competitive relationship between these commensals and C. difficile using an in vitro co-culture system. We found that inhibition of C. difficile growth by commensal Clostridia supplemented with CA was strain-dependent, correlated with the production of ∼2 mM DCA, and increased expression of bai operon genes. We also found that C. difficile was able to outcompete all four commensal Clostridia in an in vitro co-culture system. These studies are instrumental in understanding the relationship between commensal Clostridia and C. difficile in the gut, which is vital for designing targeted bacterial therapeutics. Future studies dissecting the regulation of the bai operon in vitro and in vivo and how this affects CDI will be important.ImportanceCommensal Clostridia encoding the bai operon such as C. scindens have been associated with protection against CDI, however the mechanism for this protection is unknown. Herein, we show four commensal Clostridia that encode the bai operon effect C. difficile growth in a strain-dependent manner, with and without the addition of cholate. Inhibition of C. difficile by commensals correlated with the efficient conversion of cholate to deoxycholate, a secondary bile acid that inhibits C. difficile germination, growth, and toxin production. Competition studies also revealed that C. difficile was able to outcompete the commensals in an in vitro co-culture system. These studies are instrumental in understanding the relationship between commensal Clostridia and C. difficile in the gut, which is vital for designing targeted bacterial therapeutics.}, author={Reed, A.D. and Nethery, M.A. and Stewart, A. and Barrangou, R. and Theriot, C.M.}, year={2020}, month={Jan} }
 @article{monte_nethery_barrangou_landgraf_fedorka-cray_2021, title={Whole-genome sequencing analysis and CRISPR genotyping of rare antibiotic-resistant Salmonella enterica serovars isolated from food and related sources}, volume={93}, ISSN={["1095-9998"]}, DOI={10.1016/j.fm.2020.103601}, abstractNote={For decades, Salmonella Typhimurium and Salmonella Enteritidis have prevailed in several countries as agents of salmonellosis outbreaks. In Brazil, the largest exporter of poultry meat, relatively little attention has been paid to infrequent serovars. Here, we report the emergence and characterization of rare serovars isolated from food and related sources collected between 2014 and 2016 in Brazil. Twenty-two Salmonella enterica isolates were analyzed through the use of whole-genome sequencing (WGS) and clustered regularly interspaced short palindromic repeats (CRISPR) genotyping. These isolates were classified into 10 infrequent serovars, including S. Abony, S. Isangi, S. Rochdale, S. Saphra, S. Orion, S. Ouakam, S. Grumpensis, S. Carrau, S. Abaetetuba, and S. Idikan. The presence of six antimicrobial resistance (AMR) genes, qnrB19, bla CMY-2 , tetA, aac(6')-Iaa, sul2 and fosA7, which encode resistance to quinolones, third-generation cephalosporin, tetracycline, aminoglycoside, sulfonamide and fosfomycin, respectively, were confirmed by WGS. All S. Isangi harbored qnrB19 with conserved genomic context across strains, while S. Abony harbored bla CMY-2 . Twelve (54.5%) strains displayed chromosomal mutations in parC (Thr57→Ser). Most serovars were classified as independent lineages, except S. Abony and S. Abaetetuba, which phylogenetically nested with Salmonella strains from different countries. CRISPR analysis revealed that the spacer content was strongly correlated with serovar and multi-locus sequence type for all strains, independently confirming the observed phylogenetic patterns, and highlighting the value of CRISPR-based genotyping for Salmonella. These findings add valuable information to the epidemiology of S. enterica in Brazil, where the emergency of antibiotic-resistant Salmonella continues to evolve.}, journal={FOOD MICROBIOLOGY}, author={Monte, Daniel F. M. and Nethery, Matthew A. and Barrangou, Rodolphe and Landgraf, Mariza and Fedorka-Cray, Paula J.}, year={2021}, month={Feb} }
 @article{brandt_barrangou_2019, title={Applications of CRISPR Technologies Across the Food Supply Chain}, volume={10}, ISSN={["1941-1413"]}, DOI={10.1146/annurev-food-032818-121204}, abstractNote={The food industry faces a 2050 deadline for the advancement and expansion of the food supply chain to support the world's growing population. Improvements are needed across crops, livestock, and microbes to achieve this goal. Since 2005, researchers have been attempting to make the necessary strides to reach this milestone, but attempts have fallen short. With the introduction of clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins, the food production field is now able to achieve some of its most exciting advancements since the Green Revolution. This review introduces the concept of applying CRISPR-Cas technology as a genome-editing tool for use in the food supply chain, focusing on its implementation to date in crop, livestock, and microbe production, advancement of products to market, and regulatory and societal hurdles that need to be overcome.}, number={1}, journal={ANNUAL REVIEW OF FOOD SCIENCE AND TECHNOLOGY, VOL 10}, publisher={Annual Reviews}, author={Brandt, Katelyn and Barrangou, Rodolphe}, year={2019}, pages={133–150} }
 @article{foley_o'flaherty_barrangou_theriot_2019, title={Bile salt hydrolases: Gatekeepers of bile acid metabolism and host-microbiome crosstalk in the gastrointestinal tract}, volume={15}, ISSN={["1553-7374"]}, url={https://doi.org/10.1371/journal.ppat.1007581}, DOI={10.1371/journal.ppat.1007581}, abstractNote={Research on bile acids has increased dramatically due to recent studies demonstrating their ability to significantly impact the host, microbiome, and various disease states [1–3]. Although these liver-synthesized molecules assist in the absorption and digestion of dietary fat in the intestine, their reabsorption and recirculation also gives them access to peripheral organs [4] (Fig 1A). Bile acids serve as substrates for bile acid receptors (BARs) found throughout the body that control critical regulatory and metabolic processes and therefore represent an important class of bioactive molecules [5]. Despite the importance of bile acids to host health, there remain gaps in our knowledge about the bacterial enzymes driving their composition and modification. 
 
 
 
 
 
 
Open in a separate window 
 
 
Fig 1 
 
Bile salt hydrolases act on circulating conjugated bile acids in the gut-liver axis. 
(A) Bile acids synthesized in the liver and stored in the gall bladder enter the small intestine through the duodenum where they reach millimolar concentrations. The majority of bile acids (95%) are reabsorbed in the ileum and recirculate to the liver through the portal vein. The remaining population transit to the colon as they continue to be reabsorbed, and a small (<5%) amount exit through the feces. Recirculating bile acids access host tissues outside the intestines to impart systemic effects on host physiology. (B) BSHs cleave the amide bond in conjugated bile acids to open up the bile acid pool to increased complexity. The gut microbiota performs additional chemistry on deconjugated bile acids to generate the secondary bile acid pool, which can undergo enterohepatic circulation and be reconjugated in the liver. These transformations are illustrated to the right as conjugated CA is deconjugated, subjected to 7 α-dehydroxylation to become DCA, and subsequently reconjugated. (C) Monomeric BSH overlay from Bifidobacterium longum (PDB ID 2HEZ), Enteroccocus faecalis (PDB ID 4WL3), Lactobacillus salivarius (PDB ID 5HKE), and Clostridium perfringens (PDB ID 2BJF). Hydrolyzed TDCA in the CpBSH active site is coordinated by several loops that contain the most variation in the peptide backbone compared to the other structures. BSH, bile salt hydrolase; CA, cholic acid; CpBSH, C. perfringens BSH; DCA,; TDCA, taurodeoxycholic acid; PDB ID, Protein Data Bank ID.}, number={3}, journal={PLOS PATHOGENS}, author={Foley, Matthew H. and O'Flaherty, Sarah and Barrangou, Rodolphe and Theriot, Casey M.}, editor={Knoll, Laura J.Editor}, year={2019}, month={Mar} }
 @article{barrangou_2019, title={Bringing CRISPR to the Cinema}, volume={2}, DOI={10.1089/crispr.2019.29070.rba}, abstractNote={The CRISPR JournalVol. 2, No. 4 EditorialBringing CRISPR to the CinemaRodolphe BarrangouRodolphe BarrangouEditor in Chief, The CRISPR Journal.Search for more papers by this authorPublished Online:16 Aug 2019https://doi.org/10.1089/crispr.2019.29070.rbaAboutSectionsView articleView Full TextPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail View article"Bringing CRISPR to the Cinema." The CRISPR Journal, 2(4), p. 187FiguresReferencesRelatedDetails Volume 2Issue 4Aug 2019 InformationCopyright 2019, Mary Ann Liebert, Inc., publishersTo cite this article:Rodolphe Barrangou.Bringing CRISPR to the Cinema.The CRISPR Journal.Aug 2019.187-187.http://doi.org/10.1089/crispr.2019.29070.rbaPublished in Volume: 2 Issue 4: August 16, 2019PDF download}, number={4}, journal={The CRISPR Journal}, publisher={Mary Ann Liebert Inc}, author={Barrangou, Rodolphe}, year={2019}, month={Aug}, pages={187–187} }
 @article{barrangou_2019, title={CRISPR on the Move in 2019}, volume={2}, DOI={10.1089/crispr.2019.29043.rba}, abstractNote={The CRISPR JournalVol. 2, No. 1 EditorialCRISPR on the Move in 2019Rodolphe BarrangouRodolphe BarrangouDr. Barrangou is a co-founder of Intellia Therapeutics and Locus Biosciences.Editor-in-Chief, The CRISPR JournalSearch for more papers by this authorPublished Online:21 Feb 2019https://doi.org/10.1089/crispr.2019.29043.rbaAboutSectionsView articleView Full TextPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail View article"CRISPR on the Move in 2019." The CRISPR Journal, 2(1), pp. 1–2FiguresReferencesRelatedDetails Volume 2Issue 1Feb 2019 InformationCopyright 2019, Mary Ann Liebert, Inc., publishersTo cite this article:Rodolphe Barrangou.CRISPR on the Move in 2019.The CRISPR Journal.Feb 2019.1-2.http://doi.org/10.1089/crispr.2019.29043.rbaPublished in Volume: 2 Issue 1: February 21, 2019PDF download}, number={1}, journal={The CRISPR Journal}, publisher={Mary Ann Liebert Inc}, author={Barrangou, Rodolphe}, year={2019}, month={Feb}, pages={1–2} }
 @misc{barrangou_notebaart_2019, title={CRISPR-Directed Microbiome Manipulation across the Food Supply Chain}, volume={27}, ISSN={["1878-4380"]}, url={https://doi.org/10.1016/j.tim.2019.03.006}, DOI={10.1016/j.tim.2019.03.006}, abstractNote={The advent of CRISPR-based technologies has revolutionized genetics over the past decade, and genome editing is now widely implemented for diverse medical and agricultural applications, such as correcting genetic disorders and improving crop and livestock breeding. CRISPR-based technologies are also of great potential to alter the genetic content of food bacteria in order to control the composition and activity of microbial populations across the food supply chain, from the farm to consumer products. Advancing the food supply chain is of great societal importance as it involves optimizing fermentation processes to enhance taste and sensory properties of food products, as well as improving food quality and safety by controlling spoilage bacteria and pathogens. Here, we discuss the various CRISPR technologies that can alter bacterial functionalities and modulate the composition of microbial communities in foods. We illustrate how these applications can be harnessed along the food supply chain to manipulate microbiomes that encompass spoilage and pathogenic bacteria as well as desirable starter cultures and health-promoting probiotics.}, number={6}, journal={TRENDS IN MICROBIOLOGY}, publisher={Elsevier BV}, author={Barrangou, Rodolphe and Notebaart, Richard A.}, year={2019}, month={Jun}, pages={489–496} }
 @article{huang_porter_zhang_barrangou_2019, title={Collaborative networks in gene editing}, volume={37}, ISSN={["1546-1696"]}, DOI={10.1038/s41587-019-0275-z}, number={10}, journal={NATURE BIOTECHNOLOGY}, publisher={Springer Science and Business Media LLC}, author={Huang, Ying and Porter, Alan and Zhang, Yi and Barrangou, Rodolphe}, year={2019}, month={Oct}, pages={1107–1109} }
 @article{nethery_henriksen_daughtry_johanningsmeier_barrangou_2019, title={Comparative genomics of eight Lactobacillus buchneri strains isolated from food spoilage}, volume={20}, ISSN={["1471-2164"]}, url={https://doi.org/10.1186/s12864-019-6274-0}, DOI={10.1186/s12864-019-6274-0}, abstractNote={
                Abstract
              
                Background
                Lactobacillus buchneri is a lactic acid bacterium frequently associated with food bioprocessing and fermentation and has been found to be either beneficial or detrimental to industrial food processes depending on the application. The ability to metabolize lactic acid into acetic acid and 1,2-propandiol makes L. buchneri invaluable to the ensiling process, however, this metabolic activity leads to spoilage in other applications, and is especially damaging to the cucumber fermentation industry. This study aims to augment our genomic understanding of L. buchneri in order to make better use of the species in a wide range of applicable industrial settings.
              
                Results
                Whole-genome sequencing (WGS) was performed on seven phenotypically diverse strains isolated from spoiled, fermented cucumber and the ATCC type strain for L. buchneri, ATCC 4005. Here, we present our findings from the comparison of eight newly-sequenced and assembled genomes against two publicly available closed reference genomes, L. buchneri CD034 and NRRL B-30929. Overall, we see ~ 50% of all coding sequences are conserved across these ten strains. When these coding sequences are clustered by functional description, the strains appear to be enriched in mobile genetic elements, namely transposons. All isolates harbor at least one CRISPR-Cas system, and many contain putative prophage regions, some of which are targeted by the host’s own DNA-encoded spacer sequences.
              
                Conclusions
                Our findings provide new insights into the genomics of L. buchneri through whole genome sequencing and subsequent characterization of genomic features, building a platform for future studies and identifying elements for potential strain manipulation or engineering.
              }, number={1}, journal={BMC GENOMICS}, publisher={Springer Science and Business Media LLC}, author={Nethery, Matthew A. and Henriksen, Emily DeCrescenzo and Daughtry, Katheryne V and Johanningsmeier, Suzanne D. and Barrangou, Rodolphe}, year={2019}, month={Nov} }
 @misc{makarova_wolf_iranzo_shmakov_alkhnbashi_brouns_charpentier_cheng_haft_horvath_et al._2020, title={Evolutionary classification of CRISPR-Cas systems: a burst of class 2 and derived variants}, volume={18}, ISSN={["1740-1534"]}, DOI={10.1038/s41579-019-0299-x}, abstractNote={The number and diversity of known CRISPR–Cas systems have substantially increased in recent years. Here, we provide an updated evolutionary classification of CRISPR–Cas systems and cas genes, with an emphasis on the major developments that have occurred since the publication of the latest classification, in 2015. The new classification includes 2 classes, 6 types and 33 subtypes, compared with 5 types and 16 subtypes in 2015. A key development is the ongoing discovery of multiple, novel class 2 CRISPR–Cas systems, which now include 3 types and 17 subtypes. A second major novelty is the discovery of numerous derived CRISPR–Cas variants, often associated with mobile genetic elements that lack the nucleases required for interference. Some of these variants are involved in RNA-guided transposition, whereas others are predicted to perform functions distinct from adaptive immunity that remain to be characterized experimentally. The third highlight is the discovery of numerous families of ancillary CRISPR-linked genes, often implicated in signal transduction. Together, these findings substantially clarify the functional diversity and evolutionary history of CRISPR–Cas. The number and diversity of known CRISPR–Cas systems have substantially increased in recent years. In this Review, Koonin and colleagues provide an updated evolutionary classification of CRISPR–Cas systems and cas genes, with an emphasis on major developments, and outline a complete scenario for the origins and evolution of CRISPR–Cas systems.}, number={2}, journal={NATURE REVIEWS MICROBIOLOGY}, author={Makarova, Kira S. and Wolf, Yuri I and Iranzo, Jaime and Shmakov, Sergey A. and Alkhnbashi, Omer S. and Brouns, Stan J. J. and Charpentier, Emmanuelle and Cheng, David and Haft, Daniel H. and Horvath, Philippe and et al.}, year={2020}, month={Feb}, pages={67–83} }
 @article{barrangou_2019, title={Foresight is 2020: Ten Bold Predictions for the New CRISPR Year}, volume={2}, DOI={10.1089/crispr.2019.29075.rba}, abstractNote={The CRISPR JournalVol. 2, No. 6 EditorialsFree AccessForesight is 2020: Ten Bold Predictions for the New CRISPR YearRodolphe BarrangouRodolphe BarrangouEditor-in-Chief, The CRISPR JournalSearch for more papers by this authorPublished Online:16 Dec 2019https://doi.org/10.1089/crispr.2019.29075.rbaAboutSectionsPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail Whereas most people use the last few days of the year to reflect on the events that shaped the previous 12 months, I have decided to embrace the disruptiveness of CRISPR and pre-emptively offer 10 bold predictions to set the stage for what promises to be yet another eventful, perhaps pivotal, year for genome editing.Rather than offer a linear progression of incremental insights into what is likely to come, I will channel the ability of our field to leap ahead and offer colorful, perhaps contrarian, predictions of what may (hopefully will) happen for CRISPR in the next 12 months.1.Notwithstanding lingering technical issues (e.g., off-target effects and immunogenicity concerns) and public apprehension, there will be several genome editing clinical successes. This year closes with promising early clinical news from Victoria Gray, the first U.S.–based sickle-cell patient treated with a CRISPR therapy. In 2020, investigational new drug filings will trend up and U.S. FDA regulators will support active recruiting for several clinical trials. I hope they will quickly generate positive results for several indications by various groups of investigators and clinicians. Both safety and efficacy for multiple indications may well be in the cards for 2020.2.In an era of scientific skepticism, the public will embrace CRISPR and increasingly appreciate the real-world benefits of genome editing. Despite widespread misinformation, distrust in the scientific enterprise, and low confidence in scientists dedicating their lives to solving real challenges, team science will step up and share more wonderful stories. In place of sensational media headlines and overdramatized fearmongering, I am looking for the real stories of the scientists leading the revolution and of the people benefiting from it.3.Europe catches up with the world. The good thing about science is that (sometimes) it is immune to politics—just sound thinking, creative minds, and the scientific method. European pundits and regulators will eventually open their eyes to see the light and objectively assess the upside of CRISPR for diverse applications for their health, food, environmental, and commercial benefits. #crispEUr4.Rather than continue with toolbox expansion, current CRISPR tools will be put to good use. After years of next-generation Cas mining and optimization of the Cas, dCas, nCas and fused-effector domains, we will appreciate that the current tools are polished enough for most users and uses. With a useful Cas (Cas-9, 12, 13, et al.) toolbox, diverse applications (editing the genome, transcriptome, and epigenome) and optimized technologies (such as prime editing), currently available tools will be harnessed and implemented with greater urgency and less concerns about technology enhancement.5.Beyond therapeutics. With increasingly promising signs of clinical success, the potential of genome editing will be unleashed for livestock, crops, and even trees for a more sustainable agriculture and healthier planet. Organisms spanning most branches of the tree of life will be enhanced for broad societal benefits.6.Cooler heads prevail. Despite the continuation of intellectual property disputes and interference proceedings, progress toward commercialization of actual products and the need to split large pies that cannot be eaten whole will compel dominant parties to partner and split the proceeds. After all, science is a team sport hinging on scientific collaborations, and the community spirit will behoove key players and leaders to play nice.7.Business dealmakers join the fray. Investors and strategists will be intrigued by deflated stock prices given the extraordinary potential. Underperforming CRISPR stocks and underwhelming financial performance—symptoms that have affected the biotech sector as a whole—have not dampened the upside of CRISPR. The recent report of sickle-cell clinical data pushed valuations significantly higher. It would not be a surprise to see pharma competitively bid for early-stage CRISPR companies and undervalued tickers. Will we see our first CRISPR start-up acquisition in 2020? #CRISPRM&A8.CRISPR responsibility. Two major reports on germline editing, from the National Academies/Royal Society and the World Health Organization, will be released in 2020. We hope the reports will coordinate, with all the voices of CRISPR being heard, so we can build consensual and broadly acceptable frameworks to ensure we use CRISPR responsibly, especially regarding usage in human embryos for germline editing. The public has asked for it, and the community has been working on it. The science versus society gap will be bridged.9.CRISPR fatigue. Despite all the fanfare, I suspect there will be some CRISPR fatigue in 2020: after years of Addgene-fueled democratization, Odin-fed biohacking, and inexorable publication and citation growth, the rate at which CRISPR is expanding will start to slow down and plateau. This is not necessarily a bad thing, as most users in need have already adopted this technology.10.CRISPR goes global. Beyond academic scientists blazing new trails and investors hunting for new technologies, nations will define visions and strategies to expand and build national CRISPR portfolios to harness the bio-economy and keep up with the competition. Leading nations have claimed a stake in the scientific literature and intellectual property arenas, but as business appetite broadens and commercialization success advances, it will be perilous not to seize editing opportunities.So get some rest to prepare for an exciting and eventful year ahead!FiguresReferencesRelatedDetails Volume 2Issue 6Dec 2019 InformationCopyright 2019, Mary Ann Liebert, Inc., publishersTo cite this article:Rodolphe Barrangou.Foresight is 2020: Ten Bold Predictions for the New CRISPR Year.The CRISPR Journal.Dec 2019.341-342.http://doi.org/10.1089/crispr.2019.29075.rbaPublished in Volume: 2 Issue 6: December 16, 2019PDF download}, number={6}, journal={The CRISPR Journal}, publisher={Mary Ann Liebert Inc}, author={Barrangou, Rodolphe}, year={2019}, pages={341–342} }
 @article{hidalgo-cantabrana_goh_pan_sanozky-dawes_barrangou_2019, title={Genome editing using the endogenous type I CRISPR-Cas system in Lactobacillus crispatus}, url={https://doi.org/10.1073/pnas.1905421116}, DOI={10.1073/pnas.1905421116}, abstractNote={
            CRISPR-Cas systems are now widely used for genome editing and transcriptional regulation in diverse organisms. The compact and portable nature of class 2 single effector nucleases, such as Cas9 or Cas12, has facilitated directed genome modifications in plants, animals, and microbes. However, most CRISPR-Cas systems belong to the more prevalent class 1 category, which hinges on multiprotein effector complexes. In the present study, we detail how the native type I-E CRISPR-Cas system, with a 5′-AAA-3′ protospacer adjacent motif (PAM) and a 61-nucleotide guide CRISPR RNA (crRNA) can be repurposed for efficient chromosomal targeting and genome editing in
            Lactobacillus crispatus
            , an important commensal and beneficial microbe in the vaginal and intestinal tracts. Specifically, we generated diverse mutations encompassing a 643-base pair (bp) deletion (100% efficiency), a stop codon insertion (36%), and a single nucleotide substitution (19%) in the exopolysaccharide priming-glycosyl transferase (
            p-gtf
            ). Additional genetic targets included a 308-bp deletion (20%) in the prophage DNA packaging
            Nu1
            and a 730-bp insertion of the green fluorescent protein gene downstream of enolase (23%). This approach enables flexible alteration of the formerly genetically recalcitrant species
            L. crispatus
            , with potential for probiotic enhancement, biotherapeutic engineering, and mucosal vaccine delivery. These results also provide a framework for repurposing endogenous CRISPR-Cas systems for flexible genome targeting and editing, while expanding the toolbox to include one of the most abundant and diverse systems found in nature.
          }, journal={Proceedings of the National Academy of Sciences}, author={Hidalgo-Cantabrana, Claudio and Goh, Yong Jun and Pan, Meichen and Sanozky-Dawes, Rosemary and Barrangou, Rodolphe}, year={2019}, month={Aug} }
 @article{canez_selle_goh_barrangou_2019, title={Outcomes and characterization of chromosomal self-targeting by native CRISPR-Cas systems in Streptococcus thermophilus}, volume={366}, ISSN={["1574-6968"]}, url={https://doi.org/10.1093/femsle/fnz105}, DOI={10.1093/femsle/fnz105}, abstractNote={ABSTRACT
               CRISPR-Cas systems provide adaptive immunity against phages in prokaryotes via DNA-encoded, RNA-mediated, nuclease-dependent targeting and cleavage. Due to inefficient and relatively limited DNA repair pathways in bacteria, CRISPR-Cas systems can be repurposed for lethal DNA targeting that selects for sequence variants. In this study, the relative killing efficiencies of endogenous Type I and Type II CRISPR-Cas systems in the model organism Streptococcus thermophilus DGCC7710 were assessed. Additionally, the genetic and phenotypic outcomes of chromosomal targeting by plasmid-programmed Type I-E or Type II-A systems were analyzed. Efficient killing was observed using both systems, in a dose-dependent manner when delivering 0.4–400 ng of plasmid DNA. Targeted PCR screening and genome sequencing were used to determine the genetic basis enabling survival, showing that evasion of Type I-E self-targeting was primarily the result of low-frequency defective plasmids that excised the targeting spacer. The most notable genotype recovered from Type II-A targeting of genomic locus, lacZ, was a 34 kb-deletion derived from homologous recombination (HR) between identical conserved sequences in two separate galE coding regions, resulting in 2% loss of the genome. Collectively, these results suggest that HR contributes to the plasticity and remodeling of bacterial genomes, leading to evasion of genome targeting by CRISPR-Cas systems.}, number={9}, journal={FEMS MICROBIOLOGY LETTERS}, publisher={Oxford University Press (OUP)}, author={Canez, Cassandra and Selle, Kurt and Goh, Yong Jun and Barrangou, Rodolphe}, year={2019}, month={May} }
 @article{barrangou_2019, title={Partnering with bioRxiv}, volume={2}, DOI={10.1089/crispr.2019.29076.rba}, abstractNote={The CRISPR JournalVol. 2, No. 6 EditorialsPartnering with bioRxivRodolphe BarrangouRodolphe BarrangouSearch for more papers by this authorPublished Online:16 Dec 2019https://doi.org/10.1089/crispr.2019.29076.rbaAboutSectionsView articleView Full TextPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail View article"Partnering with bioRxiv." The CRISPR Journal, 2(6), p. 342FiguresReferencesRelatedDetails Volume 2Issue 6Dec 2019 InformationCopyright 2019, Mary Ann Liebert, Inc., publishersTo cite this article:Rodolphe Barrangou.Partnering with bioRxiv.The CRISPR Journal.Dec 2019.342-342.http://doi.org/10.1089/crispr.2019.29076.rbaPublished in Volume: 2 Issue 6: December 16, 2019PDF download}, number={6}, journal={The CRISPR Journal}, publisher={Mary Ann Liebert Inc}, author={Barrangou, Rodolphe}, year={2019}, pages={342–342} }
 @article{davis_2019, title={Profile of Rodolphe Barrangou}, volume={116}, DOI={10.1073/pnas.1911079116}, abstractNote={CRISPR, the Instapot of genome editing tools, has its origins in a bacterial immune system that recognizes and slices the genetic material of invading phages. Rodolphe Barrangou, a professor of food science at North Carolina State University, demonstrated the original function of the characteristic repeating genetic sequences long before it became a household word. Barrangou is now turning CRISPR inward, using bacterial cells’ own machinery to edit bacteria. “Unfortunately, bacteria do not typically have good DNA repair mechanisms, so self-targeting usually turns out to be lethal,” explains Barrangou, who was elected to the National Academy of Sciences in 2018. In his Inaugural Article, Barrangou outlines how repurposing the existing type I-E CRISPR-Cas3 system of Lactobacillus crispatus and inserting repair templates can enable targeted editing of this common member of the human microbiome (1).



Photograph of Rodolphe Barrangou. Image courtesy of North Carolina State University/Marc Hall.





Probiotic bacterium L. acidophilus NCFM. Image courtesy of North Carolina State University/Courtney Klotz, Valerie Lapham, and Charles Mooney.



Born in France in 1975, Barrangou found his appetite for science relatively late, when he decided to major in chemistry at the Universite Rene Descartes in Paris. After earning a bachelor’s degree in 1996, he pursued a master’s degree in biological engineering at the Universite de Technologie Compiegne. Barrangou could not envision spending “10 to 20 years working on 1 molecule, 1 project,” as an organic chemist, and engineering was not quite the right fit either. A microbiology class on fermentation propelled him toward a second master’s degree in food science and the field he has helped shape for the past 2 decades. “The living part of microbes was a whole different dimensionality.”}, number={32}, journal={Proceedings of the National Academy of Sciences}, publisher={Proceedings of the National Academy of Sciences}, author={Davis, Tinsley H.}, year={2019}, month={Jul}, pages={15754–15756} }
 @article{varble_meaden_barrangou_westra_marraffini_2019, title={Recombination between phages and CRISPR-cas loci facilitates horizontal gene transfer in staphylococci}, volume={4}, ISSN={["2058-5276"]}, url={https://doi.org/10.1038/s41564-019-0400-2}, DOI={10.1038/s41564-019-0400-2}, abstractNote={CRISPR (clustered regularly interspaced short palindromic repeats) loci and their associated (cas) genes encode an adaptive immune system that protects prokaryotes from viral1 and plasmid2 invaders. Following viral (phage) infection, a small fraction of the prokaryotic cells are able to integrate a small sequence of the invader's genome into the CRISPR array1. These sequences, known as spacers, are transcribed and processed into small CRISPR RNA guides3–5 that associate with Cas nucleases to specify a viral target for destruction6–9. Although CRISPR−cas loci are widely distributed throughout microbial genomes and often display hallmarks of horizontal gene transfer10–12, the drivers of CRISPR dissemination remain unclear. Here, we show that spacers can recombine with phage target sequences to mediate a form of specialized transduction of CRISPR elements. Phage targets in phage 85, ΦNM1, ΦNM4 and Φ12 can recombine with spacers in either chromosomal or plasmid-borne CRISPR loci in Staphylococcus, leading to either the transfer of CRISPR-adjacent genes or the propagation of acquired immunity to other bacteria in the population, respectively. Our data demonstrate that spacer sequences not only specify the targets of Cas nucleases but also can promote horizontal gene transfer. CRISPR spacers can recombine with phage target sequences to mediate a form of specialized transduction that can promote transfer of CRISPR elements to other bacteria in the population.}, number={6}, journal={NATURE MICROBIOLOGY}, publisher={Springer Nature}, author={Varble, Andrew and Meaden, Sean and Barrangou, Rodolphe and Westra, Edze R. and Marraffini, Luciano A.}, year={2019}, month={Jun}, pages={956–963} }
 @article{selle_andersen_barrangou_2019, title={Short communication: Transcriptional response to a large genomic island deletion in the dairy starter culture Streptococcus thermophilus}, volume={102}, ISSN={["1525-3198"]}, DOI={10.3168/jds.2019-16397}, abstractNote={Streptococcus thermophilus is a lactic acid bacterium widely used in the syntrophic fermentation of milk into yogurt and cheese. Streptococcus thermophilus has adapted to ferment milk primarily through reductive genome evolution but also through acquisition of genes conferring proto-cooperation with Lactobacillus bulgaricus and efficient metabolism of milk macronutrients. Genomic analysis of Strep. thermophilus strains suggests that mobile genetic elements have contributed to genomic evolution through horizontal gene transfer and genomic plasticity. We previously used the endogenous type II CRISPR-Cas [clustered regularly interspaced short palindromic repeats (CRISPR) with CRISPR-associated sequences (Cas)] system in Strep. thermophilus to isolate derivatives lacking the chromosomal mobile genetic element and expandable island that display decreased fitness under routine culturing conditions. Of note, the Lac operon and Leloir pathway genes were deleted in the largest expendable genomic island (102 kbp), rendering the strain incapable of acidifying milk. However, the removal of other open reading frames in the same island had unclear effects on the fitness and regulatory networks of Strep. thermophilus. To uncover the physiological basis for the observed phenotypic changes and underlying regulatory networks affected by deletion of the 102-kbp genomic island in Strep. thermophilus, we analyzed the transcriptome of the mutant that lacked ~5% of its genome. In addition to the loss of transcripts encoded by the deleted material, we detected a total of 56 genes that were differentially expressed, primarily encompassing 10 select operons. Several predicted metabolic pathways were affected, including amino acid and purine metabolism, oligopeptide transport, and iron transport. Collectively, these results suggest that deletion of a 102-kb genomic island in Strep. thermophilus influences compensatory transcription of starvation stress response genes and metabolic pathways involved in important niche-related adaptation.}, number={9}, journal={JOURNAL OF DAIRY SCIENCE}, publisher={American Dairy Science Association}, author={Selle, Kurt and Andersen, Joakim M. and Barrangou, Rodolphe}, year={2019}, month={Sep}, pages={7800–7806} }
 @article{barrangou_2019, title={Taking CRISPR to New Heights}, volume={2}, DOI={10.1089/crispr.2019.29064.rba}, abstractNote={The CRISPR JournalVol. 2, No. 3 EditorialTaking CRISPR to New HeightsRodolphe BarrangouRodolphe BarrangouEditor-in-Chief, The CRISPR Journal.Search for more papers by this authorPublished Online:21 Jun 2019https://doi.org/10.1089/crispr.2019.29064.rbaAboutSectionsView articleView Full TextPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail View article"Taking CRISPR to New Heights." The CRISPR Journal, 2(3), p. 133FiguresReferencesRelatedDetails Volume 2Issue 3Jun 2019 InformationCopyright 2019, Mary Ann Liebert, Inc., publishersTo cite this article:Rodolphe Barrangou.Taking CRISPR to New Heights.The CRISPR Journal.Jun 2019.133-133.http://doi.org/10.1089/crispr.2019.29064.rbaPublished in Volume: 2 Issue 3: June 21, 2019PDF download}, number={3}, journal={The CRISPR Journal}, publisher={Mary Ann Liebert Inc}, author={Barrangou, Rodolphe}, year={2019}, month={Jun}, pages={133–133} }
 @article{pickar-oliver_black_lewis_mutchnick_klann_gilcrest_sitton_nelson_barrera_bartelt_et al._2019, title={Targeted transcriptional modulation with type I CRISPR-Cas systems in human cells}, volume={37}, ISSN={["1546-1696"]}, DOI={10.1038/s41587-019-0235-7}, abstractNote={Class 2 CRISPR–Cas systems, such as Cas9 and Cas12, have been widely used to target DNA sequences in eukaryotic genomes. However, class 1 CRISPR–Cas systems, which represent about 90% of all CRISPR systems in nature, remain largely unexplored for genome engineering applications. Here, we show that class 1 CRISPR–Cas systems can be expressed in mammalian cells and used for DNA targeting and transcriptional control. We repurpose type I variants of class 1 CRISPR–Cas systems from Escherichia coli and Listeria monocytogenes, which target DNA via a multi-component RNA-guided complex termed Cascade. We validate Cascade expression, complex formation and nuclear localization in human cells, and demonstrate programmable CRISPR RNA (crRNA)-mediated targeting of specific loci in the human genome. By tethering activation and repression domains to Cascade, we modulate the expression of targeted endogenous genes in human cells. This study demonstrates the use of Cascade as a CRISPR-based technology for targeted eukaryotic gene regulation, highlighting class 1 CRISPR–Cas systems for further exploration. Type I CRISPR–Cas systems, the largest group of CRISPR systems in nature, can be repurposed for DNA targeting and gene regulation in human cells}, number={12}, journal={NATURE BIOTECHNOLOGY}, publisher={Springer Science and Business Media LLC}, author={Pickar-Oliver, Adrian and Black, Joshua B. and Lewis, Mae M. and Mutchnick, Kevin J. and Klann, Tyler S. and Gilcrest, Kylie A. and Sitton, Madeleine J. and Nelson, Christopher E. and Barrera, Alejandro and Bartelt, Luke C. and et al.}, year={2019}, month={Dec}, pages={1493-+} }
 @article{young_gasior_jones_wang_navarro_vickroy_barrangou_2019, title={The repurposing of type I-E CRISPR-Cascade for gene activation in plants}, url={https://doi.org/10.1038/s42003-019-0637-6}, DOI={10.1038/s42003-019-0637-6}, abstractNote={AbstractCRISPR-Cas systems are robust and facile tools for manipulating the genome, epigenome and transcriptome of eukaryotic organisms. Most groups use class 2 effectors, such as Cas9 and Cas12a, however, other CRISPR-Cas systems may provide unique opportunities for genome engineering. Indeed, the multi-subunit composition of class 1 systems offers to expand the number of domains and functionalities that may be recruited to a genomic target. Here we report DNA targeting in Zea mays using a class 1 type I-E CRISPR-Cas system from S. thermophilus. First, we engineer its Cascade complex to modulate gene expression by tethering a plant transcriptional activation domain to 3 different subunits. Next, using an immunofluorescent assay, we confirm Cascade cellular complex formation and observe enhanced gene activation when multiple subunits tagged with the transcriptional activator are combined. Finally, we examine Cascade mediated gene activation at chromosomal DNA targets by reprogramming Zea mays cells to change color.}, journal={Communications Biology}, author={Young, Joshua K. and Gasior, Stephen L. and Jones, Spencer and Wang, Lijuan and Navarro, Pedro and Vickroy, Becca and Barrangou, Rodolphe}, year={2019}, month={Oct} }
 @article{barrangou_2019, title={Thinking About CRISPR: The Ethics of Human Genome Editing}, volume={2}, DOI={10.1089/crispr.2019.29072.rba}, abstractNote={The CRISPR JournalVol. 2, No. 5 EditorialThinking About CRISPR: The Ethics of Human Genome EditingRodolphe BarrangouRodolphe BarrangouEditor-in-Chief, The CRISPR Journal.Search for more papers by this authorPublished Online:9 Oct 2019https://doi.org/10.1089/crispr.2019.29072.rbaAboutSectionsView articleView Full TextPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail View article"Thinking About CRISPR: The Ethics of Human Genome Editing." The CRISPR Journal, 2(5), pp. 247–248FiguresReferencesRelatedDetailsCited byDemocratizing CRISPR? Stories, practices, and politics of science and governance on the agricultural gene editing frontier25 February 2020 | Elementa: Science of the Anthropocene, Vol. 8 Volume 2Issue 5Oct 2019 InformationCopyright 2019, Mary Ann Liebert, Inc., publishersTo cite this article:Rodolphe Barrangou.Thinking About CRISPR: The Ethics of Human Genome Editing.The CRISPR Journal.Oct 2019.247-248.http://doi.org/10.1089/crispr.2019.29072.rbaPublished in Volume: 2 Issue 5: October 9, 2019PDF download}, number={5}, journal={The CRISPR Journal}, publisher={Mary Ann Liebert Inc}, author={Barrangou, Rodolphe}, year={2019}, month={Oct}, pages={247–248} }
 @article{barrangou_2019, title={Time To Let CRISPR B.E.?}, volume={2}, DOI={10.1089/crispr.2019.29055.rdb}, abstractNote={The CRISPR JournalVol. 2, No. 2 EditorialTime To Let CRISPR B.E.?Rodolphe BarrangouRodolphe BarrangouEditor-in-Chief, The CRISPR JournalSearch for more papers by this authorPublished Online:18 Apr 2019https://doi.org/10.1089/crispr.2019.29055.rdbAboutSectionsView articleView Full TextPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail View article"Time To Let CRISPR B.E.?." The CRISPR Journal, 2(2), p. 67FiguresReferencesRelatedDetails Volume 2Issue 2Apr 2019 InformationCopyright 2019, Mary Ann Liebert, Inc., publishersTo cite this article:Rodolphe Barrangou.Time To Let CRISPR B.E.?.The CRISPR Journal.Apr 2019.67-67.http://doi.org/10.1089/crispr.2019.29055.rdbPublished in Volume: 2 Issue 2: April 18, 2019PDF download}, number={2}, journal={The CRISPR Journal}, publisher={Mary Ann Liebert Inc}, author={Barrangou, Rodolphe}, year={2019}, month={Apr}, pages={67–67} }
 @article{barrangou_2018, title={CRISPR Craziness: A Response to the EU Court Ruling}, volume={1}, DOI={10.1089/crispr.2018.29025.edi}, abstractNote={The CRISPR JournalVol. 1, No. 4 EditorialsCRISPR Craziness: A Response to the EU Court RulingRodolphe BarrangouRodolphe BarrangouEditor-in-Chief, The CRISPR Journal.Search for more papers by this authorPublished Online:1 Aug 2018https://doi.org/10.1089/crispr.2018.29025.ediAboutSectionsView articleView Full TextPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail View article"CRISPR Craziness: A Response to the EU Court Ruling." The CRISPR Journal, 1(4), pp. 251–252FiguresReferencesRelatedDetailsCited byCRISPR-Cas Genome Editing for Horticultural Crops Improvement: Advantages and Prospects30 December 2022 | Horticulturae, Vol. 9, No. 1Consumer Evaluation of Novel Plant-Breeding Technologies: A Decision-Focused Research Agenda4 January 2023CRISPR and Chromothripsis: Proceed with Caution Stephanie Mack and I. Alasdair Russell16 June 2021 | The CRISPR Journal, Vol. 4, No. 3A Field Day for Gene-Edited Brassicas and Crop Improvement Johnathan A. Napier16 June 2021 | The CRISPR Journal, Vol. 4, No. 3How should we regulate products of new breeding techniques? Opinion of surveyed experts in plant biotechnologyBiotechnology Reports, Vol. 26European Court of Justice ruling regarding new genetic engineering methods scientifically justified: a commentary on the biased reporting about the recent ruling20 December 2018 | Environmental Sciences Europe, Vol. 30, No. 1 Volume 1Issue 4Aug 2018 InformationCopyright 2018, Mary Ann Liebert, Inc.To cite this article:Rodolphe Barrangou.CRISPR Craziness: A Response to the EU Court Ruling.The CRISPR Journal.Aug 2018.251-252.http://doi.org/10.1089/crispr.2018.29025.ediPublished in Volume: 1 Issue 4: August 1, 2018PDF download}, number={4}, journal={The CRISPR Journal}, publisher={Mary Ann Liebert Inc}, author={Barrangou, Rodolphe}, year={2018}, month={Aug}, pages={251–252} }
 @article{barrangou_2018, title={CRISPR Crossroads for Genome Editing}, volume={1}, DOI={10.1089/crispr.2018.29040.rba}, abstractNote={The CRISPR JournalVol. 1, No. 6 EditorialCRISPR Crossroads for Genome EditingRodolphe BarrangouRodolphe BarrangouEditor-in-Chief, The CRISPR JournalSearch for more papers by this authorPublished Online:20 Dec 2018https://doi.org/10.1089/crispr.2018.29040.rbaAboutSectionsView articleView Full TextPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail View article"CRISPR Crossroads for Genome Editing." The CRISPR Journal, 1(6), pp. 349–350FiguresReferencesRelatedDetailsCited byRealigning gene editing with clinical research ethics: What the “CRISPR Twins” debacle means for Chinese and international research ethics governance17 May 2019 | Accountability in Research, Vol. 26, No. 4 Volume 1Issue 6Dec 2018 InformationCopyright 2018, Mary Ann Liebert, Inc., publishersTo cite this article:Rodolphe Barrangou.CRISPR Crossroads for Genome Editing.The CRISPR Journal.Dec 2018.349-350.http://doi.org/10.1089/crispr.2018.29040.rbaPublished in Volume: 1 Issue 6: December 20, 2018PDF download}, number={6}, journal={The CRISPR Journal}, publisher={Mary Ann Liebert Inc}, author={Barrangou, Rodolphe}, year={2018}, pages={349–350} }
 @article{nethery_barrangou_2019, title={CRISPR Visualizer: rapid identification and visualization of CRISPR loci via an automated high-throughput processing pipeline}, volume={16}, ISSN={["1555-8584"]}, url={https://doi.org/10.1080/15476286.2018.1493332}, DOI={10.1080/15476286.2018.1493332}, abstractNote={ABSTRACT A CRISPR locus, defined by an array of repeat and spacer elements, constitutes a genetic record of the ceaseless battle between bacteria and viruses, showcasing the genomic integration of spacers acquired from invasive DNA. In particular, iterative spacer acquisitions represent unique evolutionary histories and are often useful for high-resolution bacterial genotyping, including comparative analysis of closely related organisms, clonal lineages, and clinical isolates. Current spacer visualization methods are typically tedious and can require manual data manipulation and curation, including spacer extraction at each CRISPR locus from genomes of interest. Here, we constructed a high-throughput extraction pipeline coupled with a local web-based visualization tool which enables CRISPR spacer and repeat extraction, rapid visualization, graphical comparison, and progressive multiple sequence alignment. We present the bioinformatic pipeline and investigate the loci of reference CRISPR-Cas systems and model organisms in 4 well-characterized subtypes. We illustrate how this analysis uncovers the evolutionary tracks and homology shared between various organisms through visual comparison of CRISPR spacers and repeats, driven through progressive alignments. Due to the ability to process unannotated genome files with minimal preparation and curation, this pipeline can be implemented promptly. Overall, this efficient high-throughput solution supports accelerated analysis of genomic data sets and enables and expedites genotyping efforts based on CRISPR loci.}, number={4}, journal={RNA BIOLOGY}, publisher={Informa UK Limited}, author={Nethery, Matthew A. and Barrangou, Rodolphe}, year={2019}, month={Apr}, pages={577–584} }
 @article{crawley_henriksan_barranaou_2018, title={CRISPRdisco: An Automated Pipeline for the Discovery and Analysis of CRISPR-Cas Systems}, volume={1}, ISSN={["2573-1602"]}, DOI={10.1089/crispr.2017.0022}, abstractNote={Abstract CRISPR-Cas adaptive immune systems of bacteria and archaea have catapulted into the scientific spotlight as genome editing tools. To aid researchers in the field, we have developed an automated pipeline, named CRISPRdisco (CRISPR discovery), to identify CRISPR repeats and cas genes in genome assemblies, determine type and subtype, and describe system completeness. All six major types and 23 currently recognized subtypes and novel putative V-U types are detected. Here, we use the pipeline to identify and classify putative CRISPR-Cas systems in 2,777 complete genomes from the NCBI RefSeq database. This allows comparison to previous publications and investigation of the occurrence and size of CRISPR-Cas systems. Software available at http://github.com/crisprlab/CRISPRdisco provides reproducible, standardized, accessible, transparent, and high-throughput analysis methods available to all researchers in and beyond the CRISPR-Cas research community. This tool opens new avenues to enable classification within a complex nomenclature and provides analytical methods in a field that has evolved rapidly.}, number={2}, journal={CRISPR JOURNAL}, publisher={Mary Ann Liebert Inc}, author={Crawley, Alexandra R. and Henriksan, Jams R. and Barranaou, Rodolphe}, year={2018}, month={Apr}, pages={171–181} }
 @misc{hidalgo-cantabrana_goh_barrangou_2019, title={Characterization and Repurposing of Type I and Type II CRISPR-Cas Systems in Bacteria}, volume={431}, ISSN={["1089-8638"]}, DOI={10.1016/j.jmb.2018.09.013}, abstractNote={CRISPR–Cas systems constitute the adaptive immune system of bacteria and archaea, as a sequence-specific nucleic acid targeting defense mechanism. The sequence-specific recognition and cleavage of Cas effector complexes has been harnessed to developed CRISPR-based technologies and drive the genome editing revolution underway, due to their efficacy, efficiency, and ease of implementation in a broad range of organisms. CRISPR-based technologies offer a wide variety of opportunities in genome remodeling and transcriptional regulation, opening new avenues for therapeutic and biotechnological applications. To repurpose CRISPR–Cas systems for these applications, the various elements of the system need to be first identified and functionally characterized in their native host. Bioinformatic tools are first used to identify putative CRISPR arrays and their associated genes, followed by a comprehensive characterization of the CRISPR–Cas system, encompassing predictions for guide and target sequences. Subsequently, interference assays and transcriptomic analyses should be performed to probe the functionality of the CRISPR–Cas system. Once an endogenous CRISPR–Cas system is characterized as functional, they can be readily repurposed by delivering an engineered synthetic CRISPR array or a small RNA guide for targeted gene manipulation. Alternatively, developing a plasmid-based system for heterologous expression of the necessary CRISPR components can enable exploitation in other organisms. Altogether, there is a wide diversity of native CRISPR–Cas systems in many bacteria and most archaea that await functional characterization and repurposing for genome editing applications in prokaryotes.}, number={1}, journal={JOURNAL OF MOLECULAR BIOLOGY}, publisher={Elsevier BV}, author={Hidalgo-Cantabrana, Claudio and Goh, Yong Jun and Barrangou, Rodolphe}, year={2019}, month={Jan}, pages={21–33} }
 @article{crawley_henriksen_stout_brandt_barrangou_2018, title={Characterizing the activity of abundant, diverse and active CRISPR-Cas systems in lactobacilli}, volume={8}, ISSN={2045-2322}, url={http://dx.doi.org/10.1038/S41598-018-29746-3}, DOI={10.1038/s41598-018-29746-3}, abstractNote={AbstractCRISPR-Cas systems provide immunity against phages and plasmids in bacteria and archaea. Despite the popularity of CRISPR-Cas9 based genome editing, few endogenous systems have been characterized to date. Here, we sampled 1,262 publically available lactobacilli genomes found them to be enriched with CRISPR-Cas adaptive immunity. While CRISPR-Cas is ubiquitous in some Lactobacillus species, CRISPR-Cas content varies at the strain level in most Lactobacillus species. We identified that Type II is the most abundant type across the genus, with II-A being the most dominant sub-type. We found that many Type II-A systems are actively transcribed, and encode spacers that efficiently provide resistance against plasmid uptake. Analysis of various CRISPR transcripts revealed that guide sequences are highly diverse in terms of crRNA and tracrRNA length and structure. Interference assays revealed highly diverse target PAM sequences. Lastly, we show that these systems can be readily repurposed for self-targeting by expressing an engineered single guide RNA. Our results reveal that Type II-A systems in lactobacilli are naturally active in their native host in terms of expression and efficiently targeting invasive and genomic DNA. Together, these systems increase the possible Cas9 targeting space and provide multiplexing potential in native hosts and heterologous genome editing purpose.}, number={1}, journal={Scientific Reports}, publisher={Springer Science and Business Media LLC}, author={Crawley, Alexandra B. and Henriksen, Emily D. and Stout, Emily and Brandt, Katelyn and Barrangou, Rodolphe}, year={2018}, month={Aug} }
 @article{faure_shmakov_makarova_wolf_crawley_barrangou_koonin_2019, title={Comparative genomics and evolution of trans-activating RNAs in Class 2 CRISPR-Cas systems}, volume={16}, ISSN={["1555-8584"]}, url={https://doi.org/10.1080/15476286.2018.1493331}, DOI={10.1080/15476286.2018.1493331}, abstractNote={ABSTRACT Trans-activating CRISPR (tracr) RNA is a distinct RNA species that interacts with the CRISPR (cr) RNA to form the dual guide (g) RNA in type II and subtype V-B CRISPR-Cas systems. The tracrRNA-crRNA interaction is essential for pre-crRNA processing as well as target recognition and cleavage. The tracrRNA consists of an antirepeat, which forms an imperfect hybrid with the repeat in the crRNA, and a distal region containing a Rho-independent terminator. Exhaustive comparative analysis of the sequences and predicted structures of the Class 2 CRISPR guide RNAs shows that all these guide RNAs share distinct structural features, in particular, the nexus stem-loop that separates the repeat-antirepeat hybrid from the distal portion of the tracrRNA and the conserved GU pair at that end of the hybrid. These structural constraints might ensure full exposure of the spacer for target recognition. Reconstruction of tracrRNA evolution for 4 tight bacterial groups demonstrates random drift of repeat-antirepeat complementarity within a window of hybrid stability that is, apparently, maintained by selection. An evolutionary scenario is proposed whereby tracrRNAs evolved on multiple occasions, via rearrangement of a CRISPR array to form the antirepeat in different locations with respect to the array. A functional tracrRNA would form if, in the new location, the antirepeat is flanked by sequences that meet the minimal requirements for a promoter and a Rho-independent terminator. Alternatively, or additionally, the antirepeat sequence could be occasionally ‘reset’ by recombination with a repeat, restoring the functionality of tracrRNAs that drift beyond the required minimal hybrid stability.}, number={4}, journal={RNA BIOLOGY}, publisher={Informa UK Limited}, author={Faure, Guilhem and Shmakov, Sergey A. and Makarova, Kira S. and Wolf, Yuri I. and Crawley, Alexandra B. and Barrangou, Rodolphe and Koonin, Eugene V.}, year={2019}, month={Apr}, pages={435–448} }
 @article{crawley_barrangou_2018, title={Conserved Genome Organization and Core Transcriptome of the Lactobacillus acidophilus Complex}, volume={9}, ISSN={["1664-302X"]}, DOI={10.3389/fmicb.2018.01834}, abstractNote={The Lactobacillus genus encompasses a genetically and functionally diverse group of species, and contains many strains widely formulated in the human food supply chain as probiotics and starter cultures. Within this genetically expansive group, there are several distinct clades that have high levels of homology, one of which is the Lactobacillus acidophilus group. Of the uniting features, small genomes, low GC content, adaptation to dairy environments, and fastidious growth requirements, are some of the most defining characteristics of this group. To better understand what truly links and defines this clade, we sought to characterize the genomic organization and content of the genomes of several members of this group. Through core genome analysis we explored the synteny and intrinsic genetic underpinnings of the L. acidophilus clade, and observed key features related to the evolution and adaptation of these organisms. While genetic content is able to provide a large map of the potential of each organism, it does not always reflect their functionality. Through transcriptomic data we inferred the core transcriptome of the L. acidophilus complex to better define the true metabolic capabilities that unite this clade. Using this approach we have identified seven small ORFs that are both highly conserved and transcribed in diverse members of this clade and could be potential novel small peptide or untranslated RNA regulators. Overall, our results reveal the core features of the L. acidophilus complex and open new avenues for the enhancement and formulation and of next generation probiotics and starter cultures.}, journal={FRONTIERS IN MICROBIOLOGY}, publisher={Frontiers Media SA}, author={Crawley, Alexandra B. and Barrangou, Rodolphe}, year={2018}, month={Aug} }
 @article{barrangou_2018, title={Cultivating CRISPR}, volume={1}, DOI={10.1089/crispr.2018.29011.rba}, abstractNote={The CRISPR JournalVol. 1, No. 2 EditorialCultivating CRISPRRodolphe BarrangouRodolphe BarrangouSearch for more papers by this authorPublished Online:1 Apr 2018https://doi.org/10.1089/crispr.2018.29011.rbaAboutSectionsView articleView Full TextPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail View article"Cultivating CRISPR." The CRISPR Journal, 1(2), pp. 99–100FiguresReferencesRelatedDetails Volume 1Issue 2Apr 2018 InformationCopyright 2018, Mary Ann Liebert, Inc.To cite this article:Rodolphe Barrangou.Cultivating CRISPR.The CRISPR Journal.Apr 2018.99-100.http://doi.org/10.1089/crispr.2018.29011.rbaPublished in Volume: 1 Issue 2: April 1, 2018PDF download}, number={2}, journal={The CRISPR Journal}, publisher={Mary Ann Liebert Inc}, author={Barrangou, Rodolphe}, year={2018}, month={Apr}, pages={99–100} }
 @article{stout_sanozky-dawes_goh_crawley_klaenhammer_barrangou_2018, title={Deletion-based escape of CRISPR-Cas9 targeting in Lactobacillus gasseri}, volume={164}, ISSN={["1465-2080"]}, DOI={10.1099/mic.0.000689}, abstractNote={Lactobacillus gasseri is a human commensal which carries CRISPR-Cas, an adaptive immune system that protects the cell from invasive mobile genetic elements (MGEs). However, MGEs occasionally escape CRISPR targeting due to DNA mutations that occur in sequences involved in CRISPR interference. To better understand CRISPR escape processes, a plasmid interference assay was used to screen for mutants that escape CRISPR-Cas targeting. Plasmids containing a target sequence and a protospacer adjacent motif (PAM) were transformed for targeting by the native CRISPR-Cas system. Although the primary outcome of the assay was efficient interference, a small proportion of the transformed population overcame targeting. Mutants containing plasmids that had escaped were recovered to investigate the genetic routes of escape and their relative frequencies. Deletion of the targeting spacer in the native CRISPR array was the dominant pattern of escape, accounting for 52-70 % of the mutants from two L. gasseri strains. We repeatedly observed internal deletions in the chromosomal CRISPR array, characterized by polarized excisions from the leader end that spanned 1-15 spacers, and systematically included the leader-proximal targeting spacer. This study shows that deletions of spacers within CRISPR arrays constitute a key escape mechanism to evade CRISPR targeting, while preserving the functionality of the CRISPR-Cas system. This mechanism enables cells to maintain an active immune system, but allows the uptake of potentially beneficial plasmids. Our study revealed the co-occurrence of other genomic mutations associated with various phenotypes, showing how this selection process uncovers population diversification.}, number={9}, journal={MICROBIOLOGY-SGM}, publisher={Microbiology Society}, author={Stout, Emily A. and Sanozky-Dawes, Rosemary and Goh, Yong Jun and Crawley, Alexandra B. and Klaenhammer, Todd R. and Barrangou, Rodolphe}, year={2018}, month={Sep}, pages={1098–1111} }
 @article{lamanna_barrangou_2018, title={Enabling the Rise of a CRISPR World}, volume={1}, DOI={10.1089/crispr.2018.0022}, abstractNote={Abstract CRISPR technology has dramatically changed scientists' ability to conduct research in medicine, biotechnology, and agriculture through faster, more efficient genome editing. A key driver of the technology's adoption is the easy, fast, and inexpensive access to vectors and the resulting next-generation tools by the nonprofit plasmid repository Addgene. Since 2013, Addgene has shipped over 100,000 CRISPR plasmids to more than 75 countries worldwide. This pipeline of new technologies is enabling cutting-edge research to address the grand challenges of mankind.}, number={3}, journal={The CRISPR Journal}, publisher={Mary Ann Liebert Inc}, author={LaManna, Caroline M. and Barrangou, Rodolphe}, year={2018}, month={Jun}, pages={205–208} }
 @misc{klotz_barrangou_2018, title={Engineering Components of the Lactobacillus S-Layer for Biotherapeutic Applications}, volume={9}, ISSN={["1664-302X"]}, DOI={10.3389/fmicb.2018.02264}, abstractNote={Lactic acid bacteria (LAB) are frequently harnessed for the delivery of biomolecules to mucosal tissues. Several species of Lactobacillus are commonly employed for this task, of which a subset are known to possess surface-layers (S-layers). S-layers are two-dimensional crystalline arrays of repeating proteinaceous subunits that form the outermost coating of many prokaryotic cell envelopes. Their periodicity and abundance have made them a target for numerous biotechnological applications. In the following review, we examine the multi-faceted S-layer protein (Slp), and its use in both heterologous protein expression systems and mucosal vaccine delivery frameworks, through its diverse genetic components: the strong native promoter, capable of synthesizing as many as 500 Slp subunits per second; the signal peptide that stimulates robust secretion of recombinant proteins; and the structural domains, which can be harnessed for both cell surface display of foreign peptides or adhesion enhancement of a host bacterium. Although numerous studies have established vaccine platforms based on one or more components of the Lactobacillus S-layer, this area of research still remains largely in its infancy, thus this review is meant to not only highlight past works, but also advocate for the future usage of Slps in biotherapeutic research.}, journal={FRONTIERS IN MICROBIOLOGY}, publisher={Frontiers Media SA}, author={Klotz, Courtney and Barrangou, Rodolphe}, year={2018}, month={Oct} }
 @article{barrangou_2018, title={Expanding the CRISPR Landscape on a cas by cas Basis}, volume={1}, DOI={10.1089/crispr.2018.29035.rba}, abstractNote={The CRISPR JournalVol. 1, No. 5 EditorialExpanding the CRISPR Landscape on a cas by cas BasisRodolphe BarrangouRodolphe BarrangouEditor-in-Chief, The CRISPR Journal.Search for more papers by this authorPublished Online:17 Oct 2018https://doi.org/10.1089/crispr.2018.29035.rbaAboutSectionsView articleView Full TextPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail View article"Expanding the CRISPR Landscape on a cas by cas Basis." The CRISPR Journal, 1(5), p. 303FiguresReferencesRelatedDetails Volume 1Issue 5Oct 2018 InformationCopyright 2018, Mary Ann Liebert, Inc., publishersTo cite this article:Rodolphe Barrangou.Expanding the CRISPR Landscape on a cas by cas Basis.The CRISPR Journal.Oct 2018.303-303.http://doi.org/10.1089/crispr.2018.29035.rbaPublished in Volume: 1 Issue 5: October 17, 2018PDF download}, number={5}, journal={The CRISPR Journal}, publisher={Mary Ann Liebert Inc}, author={Barrangou, Rodolphe}, year={2018}, month={Oct}, pages={303–303} }
 @misc{goh_barrangou_2019, title={Harnessing CRISPR-Cas systems for precision engineering of designer probiotic lactobacilli}, volume={56}, ISSN={["1879-0429"]}, url={https://doi.org/10.1016/j.copbio.2018.11.009}, DOI={10.1016/j.copbio.2018.11.009}, abstractNote={Our evolving understanding on the mechanisms underlying the health-promoting attributes of probiotic lactobacilli, together with an expanding genome editing toolbox have made this genus an ideal chassis for the development of living therapeutics. The rising adoption of CRISPR-based technologies for prokaryotic engineering has demonstrated precise, efficient and scalable genome editing and tunable transcriptional regulation that can be translated into next-generation development of probiotic lactobacilli with enhanced robustness and designer functionalities. Here, we discuss how these tools in conjunction with the naturally abundant and diverse native CRISPR-Cas systems can be harnessed for Lactobacillus cell surface engineering and the delivery of biotherapeutics.}, journal={CURRENT OPINION IN BIOTECHNOLOGY}, publisher={Elsevier BV}, author={Goh, Yong Jun and Barrangou, Rodolphe}, year={2019}, month={Apr}, pages={163–171} }
 @article{weissman_holmes_barrangou_moineau_fagan_levin_johnson_2018, title={Immune loss as a driver of coexistence during host-phage coevolution}, volume={12}, ISSN={["1751-7370"]}, DOI={10.1038/ismej.2017.194}, abstractNote={Abstract
               Bacteria and their viral pathogens face constant pressure for augmented immune and infective capabilities, respectively. Under this reciprocally imposed selective regime, we expect to see a runaway evolutionary arms race, ultimately leading to the extinction of one species. Despite this prediction, in many systems host and pathogen coexist with minimal coevolution even when well-mixed. Previous work explained this puzzling phenomenon by invoking fitness tradeoffs, which can diminish an arms race dynamic. Here we propose that the regular loss of immunity by the bacterial host can also produce host-phage coexistence. We pair a general model of immunity with an experimental and theoretical case study of the CRISPR-Cas immune system to contrast the behavior of tradeoff and loss mechanisms in well-mixed systems. We find that, while both mechanisms can produce stable coexistence, only immune loss does so robustly within realistic parameter ranges.}, number={2}, journal={ISME JOURNAL}, publisher={Springer Science and Business Media LLC}, author={Weissman, Jake L. and Holmes, Rayshawn and Barrangou, Rodolphe and Moineau, Sylvain and Fagan, William F. and Levin, Bruce and Johnson, Philip L. F.}, year={2018}, month={Feb}, pages={585–597} }
 @misc{hidalgo-cantabrana_sanozky-dawes_barrangou_2018, title={Insights into the Human Virome Using CRISPR Spacers from Microbiomes}, volume={10}, ISSN={["1999-4915"]}, url={https://doi.org/10.3390/v10090479}, DOI={10.3390/v10090479}, abstractNote={Due to recent advances in next-generation sequencing over the past decade, our understanding of the human microbiome and its relationship to health and disease has increased dramatically. Yet, our insights into the human virome, and its interplay with important microbes that impact human health, is relatively limited. Prokaryotic and eukaryotic viruses are present throughout the human body, comprising a large and diverse population which influences several niches and impacts our health at various body sites. The presence of prokaryotic viruses like phages, has been documented at many different body sites, with the human gut being the richest ecological niche. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and associated proteins constitute the adaptive immune system of bacteria, which prevents attack by invasive nucleic acid. CRISPR-Cas systems function by uptake and integration of foreign genetic element sequences into the CRISPR array, which constitutes a genomic archive of iterative vaccination events. Consequently, CRISPR spacers can be investigated to reconstruct interplay between viruses and bacteria, and metagenomic sequencing data can be exploited to provide insights into host-phage interactions within a niche. Here, we show how the CRISPR spacer content of commensal and pathogenic bacteria can be used to determine the evidence of their phage exposure. This framework opens new opportunities for investigating host-virus dynamics in metagenomic data, and highlights the need to dedicate more efforts for virome sampling and sequencing.}, number={9}, journal={VIRUSES-BASEL}, author={Hidalgo-Cantabrana, Claudio and Sanozky-Dawes, Rosemary and Barrangou, Rodolphe}, year={2018}, month={Sep} }
 @article{barrangou_2018, title={Keep Calm and CRISPR On}, volume={1}, ISSN={["2573-1602"]}, DOI={10.1089/crispr.2017.29000.rba}, abstractNote={The CRISPR JournalVol. 1, No. 1 EditorialKeep Calm and CRISPR OnRodolphe BarrangouRodolphe BarrangouSearch for more papers by this authorPublished Online:1 Feb 2018https://doi.org/10.1089/crispr.2017.29000.rbaAboutSectionsView articleView Full TextPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail View articleFiguresReferencesRelatedDetailsCited byDevelopment of CNN Model for Prediction of CRISPR/Cas12 Guide RNA Activity20 November 2019Breaking the germline barrier in a moral vacuum26 July 2019 | Accountability in Research, Vol. 26, No. 6Collateral damage and CRISPR genome editing14 March 2019 | PLOS Genetics, Vol. 15, No. 3How to talk about genome editing25 April 2018 | British Medical Bulletin, Vol. 126, No. 1 Volume 1Issue 1Feb 2018 InformationCopyright 2018, Mary Ann Liebert, Inc.To cite this article:Rodolphe Barrangou.Keep Calm and CRISPR On.The CRISPR Journal.Feb 2018.1-3.http://doi.org/10.1089/crispr.2017.29000.rbaPublished in Volume: 1 Issue 1: February 1, 2018Online Ahead of Print:January 8, 2018PDF download}, number={1}, journal={CRISPR JOURNAL}, publisher={Mary Ann Liebert Inc}, author={Barrangou, Rodolphe}, year={2018}, month={Feb}, pages={1–3} }
 @article{anderson_mcclelland_maksimova_strezoska_basila_briner_barrangou_smith_2018, title={Lactobacillus gasseri CRISPR-Cas9 characterization In Vitro reveals a flexible mode of protospacer-adjacent motif recognition}, volume={13}, ISSN={["1932-6203"]}, DOI={10.1371/journal.pone.0192181}, abstractNote={While the CRISPR-Cas9 system from S. pyogenes is a powerful genome engineering tool, additional programmed nucleases would enable added flexibility in targeting space and multiplexing. Here, we characterized a CRISPR-Cas9 system from L. gasseri and found that it has modest activity in a cell-free lysate assay but no activity in mammalian cells even when altering promoter, position of tag sequences and NLS, and length of crRNA:tracrRNA. In the lysate assay we tested over 400 sequential crRNA target sequences and found that the Lga Cas9 PAM is NNGA/NDRA, different than NTAA predicted from the native bacterial host. In addition, we found multiple instances of consecutive crRNA target sites, indicating flexibility in either PAM sequence or distance from the crRNA target site. This work highlights the need for characterization of new CRISPR systems and highlights the non-triviality of porting them into eukaryotes as gene editing tools.}, number={2}, journal={PLOS ONE}, publisher={Public Library of Science (PLoS)}, author={Anderson, Emily M. and McClelland, Shawn and Maksimova, Elena and Strezoska, Zaklina and Basila, Megan and Briner, Alexandra E. and Barrangou, Rodolphe and Smith, Anja van Brabant}, editor={Xu, Shuang-yongEditor}, year={2018}, month={Feb} }
 @article{davies_barrangou_2018, title={MasterChef at Work: An Interview with Rodolphe Barrangou}, volume={1}, DOI={10.1089/crispr.2018.29015.int}, abstractNote={The CRISPR JournalVol. 1, No. 3 InterviewMasterChef at Work: An Interview with Rodolphe BarrangouKevin Davies and Rodolphe BarrangouKevin DaviesSearch for more papers by this author and Rodolphe BarrangouSearch for more papers by this authorPublished Online:1 Jun 2018https://doi.org/10.1089/crispr.2018.29015.intAboutSectionsView articleView Full TextPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail View articleFiguresReferencesRelatedDetails Volume 1Issue 3Jun 2018 InformationCopyright 2018, Mary Ann Liebert, Inc.To cite this article:Kevin Davies and Rodolphe Barrangou.MasterChef at Work: An Interview with Rodolphe Barrangou.The CRISPR Journal.Jun 2018.219-222.http://doi.org/10.1089/crispr.2018.29015.intPublished in Volume: 1 Issue 3: June 1, 2018PDF download}, number={3}, journal={The CRISPR Journal}, publisher={Mary Ann Liebert Inc}, author={Davies, Kevin and Barrangou, Rodolphe}, year={2018}, month={Jun}, pages={219–222} }
 @article{barrangou_oost_2018, title={Mining for novel bacterial defence systems}, volume={3}, ISSN={["2058-5276"]}, DOI={10.1038/s41564-018-0149-z}, abstractNote={Bacteria encode many strategies to prevent or escape infection. Through the analysis of metagenomic dark matter, several novel defence systems were identified, some of which were engineered and characterized in vivo, showing that they provide resistance against viruses and plasmids.}, number={5}, journal={NATURE MICROBIOLOGY}, publisher={Springer Science and Business Media LLC}, author={Barrangou, Rodolphe and Oost, John}, year={2018}, month={May}, pages={535–536} }
 @article{daughtry_johanningsmeier_sanozky-dawes_klaenhammer_barrangou_2018, title={Phenotypic and genotypic diversity of Lactobacillus buchneri strains isolated from spoiled, fermented cucumber}, volume={280}, ISSN={["1879-3460"]}, DOI={10.1016/j.ijfoodmicro.2018.04.044}, abstractNote={Lactobacillus buchneri is a Gram-positive, obligate heterofermentative, facultative anaerobe commonly affiliated with spoilage of food products. Notably, L. buchneri is able to metabolize lactic acid into acetic acid and 1,2-propanediol. Although beneficial to the silage industry, this metabolic capability is detrimental to preservation of cucumbers by fermentation. The objective of this study was to characterize isolates of L. buchneri purified from both industrial and experimental fermented cucumber after the onset of secondary fermentation. Genotypic and phenotypic characterization included 16S rRNA sequencing, DiversiLab® rep-PCR, colony morphology, API 50 CH carbohydrate analysis, and ability to degrade lactic acid in modified MRS and fermented cucumber media. Distinct groups of isolates were identified with differing colony morphologies that varied in color (translucent white to opaque yellow), diameter (1 mm–11 mm), and shape (umbonate, flat, circular or irregular). Growth rates in MRS revealed strain differences, and a wide spectrum of carbon source utilization was observed. Some strains were able to ferment as many as 21 of 49 tested carbon sources, including inulin, fucose, gentiobiose, lactose, mannitol, potassium ketogluconate, saccharose, raffinose, galactose, and xylose, while others metabolized as few as eight carbohydrates as the sole source of carbon. All isolates degraded lactic acid in both fermented cucumber medium and modified MRS, but exhibited differences in the rate and extent of lactate degradation. Isolates clustered into eight distinct groups based on rep-PCR fingerprints with 20 of 36 of the isolates exhibiting >97% similarity. Although isolated from similar environmental niches, significant phenotypic and genotypic diversity was found among the L. buchneri cultures. A collection of unique L. buchneri strains was identified and characterized, providing the basis for further analysis of metabolic and genomic capabilities of this species to enable control of lactic acid degradation in fermented plant materials.}, journal={INTERNATIONAL JOURNAL OF FOOD MICROBIOLOGY}, publisher={Elsevier BV}, author={Daughtry, Katheryne V and Johanningsmeier, Suzanne D. and Sanozky-Dawes, Rosemary and Klaenhammer, Todd R. and Barrangou, Rodolphe}, year={2018}, month={Sep}, pages={46–56} }
 @article{curchoe_barrangou_2018, title={Pomp and Circumstance: Making the Case for CRISPR}, volume={1}, DOI={10.1089/crispr.2018.29030.oxf}, abstractNote={The CRISPR JournalVol. 1, No. 4 EditorialsPomp and Circumstance: Making the Case for CRISPRCarol Lynn Curchoe and Rodolphe BarrangouCarol Lynn CurchoeSan Diego Fertility Center and 32ATPs, San Diego, CaliforniaSearch for more papers by this author and Rodolphe BarrangouEditor-in-Chief, The CRISPR Journal.Search for more papers by this authorPublished Online:1 Aug 2018https://doi.org/10.1089/crispr.2018.29030.oxfAboutSectionsView articleView Full TextPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail View article"Pomp and Circumstance: Making the Case for CRISPR." The CRISPR Journal, 1(4), pp. 253–254FiguresReferencesRelatedDetails Volume 1Issue 4Aug 2018 InformationCopyright 2018, Mary Ann Liebert, Inc.To cite this article:Carol Lynn Curchoe and Rodolphe Barrangou.Pomp and Circumstance: Making the Case for CRISPR.The CRISPR Journal.Aug 2018.253-254.http://doi.org/10.1089/crispr.2018.29030.oxfPublished in Volume: 1 Issue 4: August 1, 2018PDF download}, number={4}, journal={The CRISPR Journal}, publisher={Mary Ann Liebert Inc}, author={Curchoe, Carol Lynn and Barrangou, Rodolphe}, year={2018}, month={Aug}, pages={253–254} }
 @article{nethery_barrangou_2019, title={Predicting and visualizing features of CRISPR-Cas systems}, volume={616}, ISSN={["0076-6879"]}, DOI={10.1016/bs.mie.2018.10.016}, abstractNote={Pervasive application of CRISPR–Cas systems in genome editing has prompted an increase in both interest and necessity to further elucidate existing systems as well as discover putative novel systems. The ubiquity and power of current computational platforms have made in silico approaches to CRISPR–Cas identification and characterization accessible to a wider audience and increasingly amenable for processing extensive data sets. Here, we describe in silico methods for predicting and visualizing notable features of CRISPR–Cas systems, including Cas domain determination, CRISPR array visualization, and inference of the protospacer-adjacent motif. The efficiency of these tools enables rapid exploration of CRISPR–Cas diversity across prokaryotic genomes and supports scalable analysis of large genomic data sets.}, journal={CRISPR-CAS ENZYMES}, author={Nethery, Matthew A. and Barrangou, Rodolphe}, year={2019}, pages={1–25} }
 @article{gersbach_barrangou_2018, title={Pulling the genome in opposite directions to dissect gene networks}, volume={19}, DOI={10.1186/s13059-018-1425-1}, abstractNote={Orthogonal CRISPR-Cas systems have been integrated into combinatorial screens to decipher complex genetic relationships in two recent studies.}, number={1}, journal={Genome Biology}, publisher={Springer Science and Business Media LLC}, author={Gersbach, Charles A. and Barrangou, Rodolphe}, year={2018}, month={Mar} }
 @article{gersbach_barrangou_2018, title={Pulling the genome in opposite directions to dissect gene networks}, volume={19}, journal={Genome Biology}, author={Gersbach, C. A. and Barrangou, R.}, year={2018} }
 @article{barrangou_2018, title={The Democratization of CRISPR}, volume={1}, DOI={10.1089/crispr.2018.29019.rba}, abstractNote={The CRISPR JournalVol. 1, No. 3 EditorialFree AccessThe Democratization of CRISPRRodolphe BarrangouRodolphe BarrangouSearch for more papers by this authorPublished Online:1 Jun 2018https://doi.org/10.1089/crispr.2018.29019.rbaAboutSectionsPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail By many measures, CRISPR*-based technologies and their applications have taken over the world in the past few years, and the CRISPR era is upon us. Much like cloning and PCR in prior decades, CRISPR stands to become a generation-defining technology. Indeed, as illustrated on the cover of this issue and discussed in the Perspective by LaManna and Barrangou on page 205, researchers continue to advance, develop, and implement a wide array of CRISPR tools, which are now being circulated globally in real time by Addgene. Embraced by commercial entities and fueled by motivated investors, the commercialization of CRISPR is next.The goal of making CRISPR openly available, regardless of location, political, or regulatory consideration, epitomizes the mission of Addgene, a nonprofit organization aimed at empowering academic laboratories by granting access to cutting-edge technologies and advance science. This is a noteworthy departure from ongoing intellectual-property disputes—and a refreshing digression from some perplexing steep licensing fees charged by not-for-profit academic institutions that hold rights to CRISPR tools.The numbers alone reflect the Addgene-enabled pervasiveness of CRISPR, not just in North America, Europe, and Asia, but also in many third world countries. CRISPR is no longer an exclusive technology but rather universal—internationally, culturally, and socially. For example, new applications of CRISPR in a diagnostic context offer great potential in territories at risk of disease outbreaks with limited resources (see the First Cut article by Karl Petri and Vikram Pattanayak, page 209). Naturally, governments and the public are actively evaluating how this technology should be managed; their opinions and actions will have widespread impact on both science and society.Public opinion has proven to be a powerful modulator of scientific progress, leading to long-lasting repercussions for many countries. As always, one hopes that scientifically informed decisions will be made, but history and recent events remind us that this is not always the case, given the public perception about science, use of misguided terms such as “frankenfood” and “designer babies” by the media, and historical shortcomings displayed by the agriculture (Ag) industry with regard to public relations, communications, and stewardship. Yet, there is hope. While much focus has been on the science, we should be mindful of the people driving these efforts. Indeed, scientists spend decades training and preparing for such opportunities and dedicate most of their lives to solving problems and creating solutions. The recent “Unite to Cure” conference at the Vatican signaled a new chapter for CRISPR acceptance and stewardship (see the First Cut by Davies on page 213) and extends a crucial dialogue about ethical implications and societal engagement.Already, CRISPR is transcending not just scientific and economic boundaries for medicine and agriculture, but also religious, ethical, and societal frontiers. Besides the well-documented therapeutic implications, notwithstanding recent Food and Drug Administration clarification requests, many signs already point to tremendous benefits pending in agriculture (see the First Cut by Willmann on page 211). Arguably, agriculture is poised to win the CRISPR race: teams of scientists in academia and industry, including “big Ag” and several new start-up companies, are harnessing next-generation breeding of crops and livestock to help feed the world. The benefits comprise classical targets such as increased yield, pest management, and drought resistance to address the food gap for our rapidly expanding population. Efforts increasingly encompass sustainability, with improved water usage and efficient land management receiving critical attention. We need more food, but also a safer and healthier supply chain, globally.Our ability to bring CRISPR to the people hinges on both supportive adjudication by regulatory agencies and legislators as well as public acceptance of science and technology. The challenges are formidable given skepticism of “big Ag” along with confusion and uncertainty over what CRISPR actually entails. Actually, there is an opportunity for both sides to discuss how to best regulate genome editing to encourage support rather than constrain the advancement and exploitation of these beneficial technologies.A major milestone was the March 2018 announcement by the U.S. Department of Agriculture stating that it will not regulate genetically edited plants that recapitulate traditional breeding results is encouraging, but this is only the beginning. Concerted efforts by multiple communities and stakeholders are underway to formalize the regulatory landscape with input from academics and PR experts. Those addressing the food gap and harnessing CRISPR-based technologies to ensure a healthier and more sustainable food supply must better convey how critical, impactful, and noble their efforts are. Unity, caution, transparency, and engagement constitute the path forward. A series of recent and forthcoming meetings will define the ability of CRISPR to make progress in the short term.For CRISPR to realize its obvious potential, the scientific community must strive to share its progress with all stakeholders: we all need to do a better job at communicating about science in general and telling and spreading the CRISPR story in particular. It is unclear if and how quickly we will get there.* Clustered Regularly Interspaced Short Palindromic Repeats.FiguresReferencesRelatedDetails Volume 1Issue 3Jun 2018 InformationCopyright 2018, Mary Ann Liebert, Inc.To cite this article:Rodolphe Barrangou.The Democratization of CRISPR.The CRISPR Journal.Jun 2018.203-204.http://doi.org/10.1089/crispr.2018.29019.rbaPublished in Volume: 1 Issue 3: June 1, 2018PDF download}, number={3}, journal={The CRISPR Journal}, publisher={Mary Ann Liebert Inc}, author={Barrangou, Rodolphe}, year={2018}, month={Jun}, pages={203–204} }
 @article{o'flaherty_crawley_theriot_barrangou_2018, title={The Lactobacillus Bile Salt Hydrolase Repertoire Reveals Niche-Specific Adaptation}, volume={3}, ISSN={["2379-5042"]}, url={https://doi.org/10.1128/mSphere.00140-18}, DOI={10.1128/msphere.00140-18}, abstractNote={
            Bile acids play an integral role in shaping the gut microbiota and host physiology by regulating metabolic signaling, weight gain, and serum cholesterol and liver triglyceride levels. Given these important roles of bile acids, we investigated the presence of bile salt hydrolase (BSH) in
            Lactobacillus
            genomes representing 170 different species, determined strain- and species-specific patterns of occurrences, and expanded on the diversity of the BSH repertoire in this genus. While our data showed that 28% of
            Lactobacillus
            species encode BSH proteins, these species are associated mainly with vertebrate-adapted niches, demonstrating selective pressure on lactobacilli to evolve to adapt to specific environments. These new data will allow targeted selection of specific strains of lactobacilli and BSH proteins for future mechanistic studies to explore their therapeutic potential for treating metabolic disorders.
          }, number={3}, journal={MSPHERE}, publisher={American Society for Microbiology}, author={O'Flaherty, Sarah and Crawley, Alexandra Briner and Theriot, Casey M. and Barrangou, Rodolphe}, editor={Ellermeier, Craig D.Editor}, year={2018} }
 @article{morovic_roos_zabel_hidalgo-cantabrana_kiefer_barrangou_2018, title={Transcriptional and Functional Analysis of Bifidobacterium animalis subsp. lactis Exposure to Tetracycline}, volume={84}, ISSN={["1098-5336"]}, url={https://doi.org/10.1128/AEM.01999-18}, DOI={10.1128/AEM.01999-18}, abstractNote={
            Bifidobacterium animalis
            subsp.
            lactis
            is widely used in human food and dietary supplements. Although well documented to be safe,
            B. animalis
            subsp.
            lactis
            strains must not contain transferable antibiotic resistance elements. Many
            B. animalis
            subsp.
            lactis
            strains have different resistance measurements despite being genetically similar, and the reasons for this are not well understood. In the current study, we sought to examine how genomic differences between two closely related industrial
            B. animalis
            subsp.
            lactis
            strains contribute to different resistance levels. This will lead to a better understanding of resistance, identify future targets for analysis of transferability, and expand our understanding of tetracycline resistance in bacteria.
          }, number={23}, journal={APPLIED AND ENVIRONMENTAL MICROBIOLOGY}, publisher={American Society for Microbiology}, author={Morovic, Wesley and Roos, Paige and Zabel, Bryan and Hidalgo-Cantabrana, Claudio and Kiefer, Anthony and Barrangou, Rodolphe}, editor={Müller, VolkerEditor}, year={2018}, month={Dec} }
 @article{brandt_barrangou_2018, title={Using glycolysis enzyme sequences to inform Lactobacillus phylogeny}, volume={4}, DOI={10.1099/mgen.0.000187}, abstractNote={The genus Lactobacillus encompasses a diversity of species that occur widely in nature and encode a plethora of metabolic pathways reflecting their adaptation to various ecological niches, including humans, animals, plants and food products. Accordingly, their functional attributes have been exploited industrially and several strains are commonly formulated as probiotics or starter cultures in the food industry. Although divergent evolutionary processes have yielded the acquisition and evolution of specialized functionalities, all Lactobacillus species share a small set of core metabolic properties, including the glycolysis pathway. Thus, the sequences of glycolytic enzymes afford a means to establish phylogenetic groups with the potential to discern species that are too closely related from a 16S rRNA standpoint. Here, we identified and extracted glycolysis enzyme sequences from 52 species, and carried out individual and concatenated phylogenetic analyses. We show that a glycolysis-based phylogenetic tree can robustly segregate lactobacilli into distinct clusters and discern very closely related species. We also compare and contrast evolutionary patterns with genome-wide features and transcriptomic patterns, reflecting genomic drift trends. Overall, results suggest that glycolytic enzymes provide valuable phylogenetic insights and may constitute practical targets for evolutionary studies.}, number={6}, journal={Microbial Genomics}, publisher={Microbiology Society}, author={Brandt, Katelyn and Barrangou, Rodolphe}, year={2018}, month={Jun} }
 @article{barrangou_horvath_2017, title={A decade of discovery: CRISPR functions and applications}, volume={2}, ISSN={["2058-5276"]}, DOI={10.1038/nmicrobiol.2017.92}, abstractNote={This year marks the tenth anniversary of the identification of the biological function of CRISPR-Cas as adaptive immune systems in bacteria. In just a decade, the characterization of CRISPR-Cas systems has established a novel means of adaptive immunity in bacteria and archaea and deepened our understanding of the interplay between prokaryotes and their environment, and CRISPR-based molecular machines have been repurposed to enable a genome editing revolution. Here, we look back on the historical milestones that have paved the way for the discovery of CRISPR and its function, and discuss the related technological applications that have emerged, with a focus on microbiology. Lastly, we provide a perspective on the impacts the field has had on science and beyond.}, number={7}, journal={NATURE MICROBIOLOGY}, publisher={Springer Nature}, author={Barrangou, Rodolphe and Horvath, Philippe}, year={2017}, month={Jul} }
 @misc{donohoue_barrangou_may_2018, title={Advances in Industrial Biotechnology Using CRISPR-Cas Systems}, volume={36}, ISSN={["1879-3096"]}, DOI={10.1016/j.tibtech.2017.07.007}, abstractNote={CRISPR-Cas systems have enabled genome editing in multiple industrially relevant species and provided genetic tools that were previously unavailable. Editing requires only two components (a Cas nuclease and a programmable guide RNA), requires minimal technical expertise to implement, and can be multiplexed for simultaneous modification of multiple sites in a single transformation event. CRISPR-Cas systems can be used to edit a genome through gene knockouts or homology-mediated knockins to control transcription of exogenous or endogenous genes, and to serve as an antimicrobial or antiviral immunization system. CRISPR-Cas-mediated engineering can increase the number of chemicals and products that are accessible through fermentation and broaden the diversity of strains suitable for industrial production. The term ‘clustered regularly interspaced short palindromic repeats’ (CRISPR) has recently become synonymous with the genome-editing revolution. The RNA-guided endonuclease CRISPR-associated protein 9 (Cas9), in particular, has attracted attention for its promise in basic research and gene editing-based therapeutics. CRISPR-Cas systems are efficient and easily programmable nucleic acid-targeting tools, with uses reaching beyond research and therapeutic development into the precision breeding of plants and animals and the engineering of industrial microbes. CRISPR-Cas systems have potential for many microbial engineering applications, including bacterial strain typing, immunization of cultures, autoimmunity or self-targeted cell killing, and the engineering or control of metabolic pathways for improved biochemical synthesis. In this review, we explore the fundamental characteristics of CRISPR-Cas systems and highlight how these features can be used in industrial settings. The term ‘clustered regularly interspaced short palindromic repeats’ (CRISPR) has recently become synonymous with the genome-editing revolution. The RNA-guided endonuclease CRISPR-associated protein 9 (Cas9), in particular, has attracted attention for its promise in basic research and gene editing-based therapeutics. CRISPR-Cas systems are efficient and easily programmable nucleic acid-targeting tools, with uses reaching beyond research and therapeutic development into the precision breeding of plants and animals and the engineering of industrial microbes. CRISPR-Cas systems have potential for many microbial engineering applications, including bacterial strain typing, immunization of cultures, autoimmunity or self-targeted cell killing, and the engineering or control of metabolic pathways for improved biochemical synthesis. In this review, we explore the fundamental characteristics of CRISPR-Cas systems and highlight how these features can be used in industrial settings. The field of industrial biology has advanced significantly in recent years due to improvements in genomic engineering techniques; specifically, improvements have emerged from altering genomic sequences via the insertion, deletion, and mutation of nucleotides, and from manipulation of the transcriptome and epigenome. Industrial biotechnology is reliant on these techniques to meet the growing demands for, and expand the catalog of, chemicals, metabolites, and biomolecules that can be produced by microbial fermentation. To date, efficient genomic engineering in microbes has relied on the use of DNA donors in combination with endogenous DNA repair machinery, exogenous recombination systems, selectable markers, site-specific recombinases (e.g., Cre-lox, FLP-FRT, etc.), group II intron retrotransposition, and the use of artificial chromosomes [1Esvelt K.M. Wang H.H. Genome-scale engineering for systems and synthetic biology.Mol. Syst. Biol. 2013; 9: 1-17Google Scholar, 2David F. Siewers V. Advances in yeast genome engineering.FEMS Yeast Res. 2015; 15: 1-14Crossref PubMed Scopus (3) Google Scholar]. Although initially impactful, such techniques are not without limitations and require the availability of selectable markers as well as multiple rounds of selection and screening to create and identify positive clones. Furthermore, they are optimized for only a handful of model microorganisms and industrial workhorse strains. The discovery and characterization of CRISPR (see Glossary) and Cas genes have fueled the recent development of a flexible, democratized genome-engineering toolbox based on programmable targeting of CRISPR-Cas systems. Researchers have applied CRISPR-Cas systems in single cell microbes to maintain genomic integrity by mitigating the effects of foreign or mobile genomic elements [3Barrangou R. et al.CRISPR provides acquired resistance against viruses in prokaryotes.Science. 2007; 315: 1709-1712Crossref PubMed Scopus (3857) Google Scholar]; to modify and manipulate genomic DNA through the introduction of double-strand breaks (DSBs), which give rise to sequence alterations when repaired by endogenous repair pathways [4Horwitz A.A. et al.Efficient multiplexed integration of synergistic alleles and metabolic pathways in yeasts via CRISPR-Cas.Cell Syst. 2015; 1: 1-9Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 5Jiang W. et al.RNA-guided editing of bacterial genomes using CRISPR-Cas systems.Nat. Biotechnol. 2013; 31: 233-239Crossref PubMed Scopus (1680) Google Scholar, 6Jiang Y. et al.Multigene editing in the Escherichia coli genome via the CRISPR-Cas9 system.Appl. Environ. 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These modes of use have direct relevance and utility in the genetic manipulation of industrial microbes where CRISPR-Cas systems can serve either to complement pre-existing techniques or to provide a new set of capabilities for organisms where tools for genetic manipulation are lacking. CRISPR-Cas systems provide endogenous adaptive immunity in approximately 40% of bacterial genomes and 70% of sequenced archaeal species [15Burstein D. et al.CRISPR-Cas viral defence systems.Nat. Commun. 2016; 7: 1-8Crossref Scopus (121) Google Scholar], and act against invading genetic elements in a conserved sequence of events: adaptation, expression, and interference (Figure 1) [16van der Oost J. et al.Unravelling the structural and mechanistic basis of CRISPR-Cas systems.Nat. Rev. Microbiol. 2015; 12: 479-492Crossref Scopus (485) Google Scholar, 17Westra E.R. et al.CRISPR-Cas systems: beyond adaptive immunity.Nat. Rev. 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CRISPR-Cas systems are genetically and mechanistically diverse, and are differentiated by the presence of either a Class 1 multiprotein effector complex (Type I, III, and IV), or a Class 2 single effector protein (Type II, V, and VI) [23Makarova K.S. et al.An updated evolutionary classification of CRISPR-Cas systems.Nat. Rev. Microbiol. 2015; 13: 722-736Crossref PubMed Scopus (1483) Google Scholar, 24Shmakov S. et al.Diversity and evolution of class 2 CRISPR–Cas systems.Nat. Rev. Microbiol. 2017; 15: 169-182Crossref PubMed Scopus (565) Google Scholar]. The various CRISPR-Cas system types are capable of DNA targeting (Type I, Type II, and V), RNA targeting (Type VI), and joint DNA and RNA targeting (Type III) [23Makarova K.S. et al.An updated evolutionary classification of CRISPR-Cas systems.Nat. Rev. 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Analysis of CRISPR arrays in S. thermophilus showed that strains that survived initial phage challenge were resistant to subsequent phage assault and had incorporated sequences from the phage genome into their CRISPR arrays [3Barrangou R. et al.CRISPR provides acquired resistance against viruses in prokaryotes.Science. 2007; 315: 1709-1712Crossref PubMed Scopus (3857) Google Scholar]. This observation led to the development of strains of S. thermophilus with a customized collection of CRISPR spacer sequences that provided immunity to common phages known to result in spoilage of industrial dairy fermentations. This initial characterization of CRISPR-Cas system immunity has informed the continued development of CRISPR applications as programmable nucleic acid-target platforms [30Garneau J.E. et al.The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA.Nature. 2010; 468: 67-71Crossref PubMed Scopus (1500) Google Scholar, 31Sapranauskas R. et al.The Streptococcus thermophilus CRISPR/Cas system provides immunity in Escherichia coli.Nucleic Acids Res. 2011; 39: 9275-9282Crossref PubMed Scopus (549) Google Scholar]. The Type II system is characterized by its signature single effector protein Cas9 [22Jinek M. et al.A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.Science. 2012; 337: 816-822Crossref PubMed Scopus (9424) Google Scholar, 32Gasiunas G. et al.Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria.Proc. Natl. Acad. Sci. U. S. 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The ability to reprogram this adaptive immune system for use across a multitude of organisms has opened new avenues for targeted genome, transcriptome, and epigenome manipulation. Since the CRISPR array serves as a genetic record of immunization events in response to exposure to invasive DNA, much like a genetic vaccination card, arrays provide insight into strain origin and divergence within a distinct bacterial species based on the collection of shared or unique spacer sequences within an array [43Timme R.E. et al.Phylogenetic diversity of the enteric pathogen Salmonella enterica subsp. enterica inferred from genome-wide reference-free SNP characters.Genome Biol. Evol. 2013; 5: 2109-2123Crossref PubMed Scopus (102) Google Scholar, 44Sheludchenko M.S. et al.CRISPR diversity in E. coli isolates from Australian animals, humans and environmental waters.PLoS One. 2015; 10: 1-12Crossref Scopus (9) Google Scholar] (Figure 2). This method of spacer oligonucleotide typing (‘spoligotyping’) [45Kamerbeek J. et al.Simultaneous detection and strain differentiation of Mycobacterium tuberculosis for diagnosis and epidemiology.J. Clin. Microbiol. 1997; 35: 907-914Crossref PubMed Google Scholar] has been used to detect, track, and type food pathogens [43Timme R.E. et al.Phylogenetic diversity of the enteric pathogen Salmonella enterica subsp. enterica inferred from genome-wide reference-free SNP characters.Genome Biol. Evol. 2013; 5: 2109-2123Crossref PubMed Scopus (102) Google Scholar], human pathogens [46Shariat, N. and Dudley, E.G. (2014) CRISPRs: molecular signatures used for pathogen subtyping. 80, 430–439.Google Scholar], and industrial starter cultures [19Horvath, P. et al. (2008) Diversity, activity, and evolution of CRISPR loci in Streptococcus thermophilus. 190, 1401–1412.Google Scholar]. Recently, Lactobacillus buchneri isolates obtained from spoiled pickle fermentations were genotyped based on spacer compositions as well as CRISPR repeat sequences, and categorized into 26 unique L. buchneri strains [47Briner A.E. Barrangou R. Lactobacillus buchneri genotyping on the basis of clustered regularly interspaced short palindromic repeat (CRISPR) locus diversity.Appl. Environ. Microbiol. 2014; 80: 994-1001Crossref PubMed Scopus (48) Google Scholar]. With further development, spoligotyping methods can enable tracking of spoilage-causing microbes back to their sources of origin (e.g., feedstock, water supply, agricultural farm, etc.) and help prevent future spoilage in industrial fermentations. As demonstrated with S. thermophilus, the adaptation, expression, and interference process of CRISPR-Cas systems can be exploited to vaccinate bacteria against potential phage threats. Bacteria with endogenous CRISPR-Cas systems can undergo repeated phage challenge to acquire spacer sequences conferring immunity [48Barrangou R. et al.Genomic impact of CRISPR immunization against bacteriophages.Biochem. Soc. Trans. 2013; 41: 1383-1391Crossref PubMed Scopus (44) Google Scholar]. Alternatively, a heterologous CRISPR-Cas system can be integrated into industrial strains to provide a synthetic immune system. Tailor-made CRISPR arrays can be preloaded with spacers targeting high-risk phage sequences, greatly reducing the time-consuming process of repetitive phage challenge and survivor recovery to create comprehensive immunity [3Barrangou R. et al.CRISPR provides acquired resistance against viruses in prokaryotes.Science. 2007; 315: 1709-1712Crossref PubMed Scopus (3857) Google Scholar]. CRISPR-Cas systems have also been used as antimicrobial selection systems through the introduction of spacers complementary to endogenous DNA sequences. In bacteria, the lack of nonhomologous end joining (NHEJ) pathways or the low expression levels of NHEJ components results in the lethality of CRISPR-Cas-mediated DNA cleavage [49Selle K. Barrangou R. Harnessing CRISPR-Cas systems for bacterial genome editing.Trends Microbiol. 2015; 23: 225-232Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar]. Several groups have tested self-targeting spacers and demonstrated sequence-specific bacterial selection [50Bikard D. et al.Development of sequence-specific antimicrobials based on programmable CRISPR-Cas nucleases.Nat. Biotechnol. 2015; 32: 1146-1150Crossref Scopus (521) Google Scholar, 51Gomaa A.A. et al.Programmable removal of bacterial strains by use of genome-targeting CRISPR-Cas Systems.MBio. 2014; 5: 1-9Crossref Scopus (248) Google Scholar, 52Citorik R.J. et al.Sequence-specific antimicrobials using efficiently delivered RNA-guided nucleases.Nat. 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Such methods of bacterial selection have potential use in industrial consortia where strain selection with antibiotics may not be feasible or is undesirable. Some studies have shown that CRISPR-Cas systems can also acquire spacers against antibiotic resistance genes and, thus, provide a means to prevent the uptake and dissemination of antibiotic-resistance cassettes [50Bikard D. et al.Development of sequence-specific antimicrobials based on programmable CRISPR-Cas nucleases.Nat. Biotechnol. 2015; 32: 1146-1150Crossref Scopus (521) Google Scholar, 56Bikard, D. et al. (2012) CRISPR interference can prevent natural transformation and virulence acquisition during in vivo bacterial infection. 12, 177–186.Google Scholar]. Alternatively, other CRISPR-Cas systems utilize a processive nuclease for interference, similar to that of the Type I Escherichia coli Cascade system with the accompanying Cas3 exonuclease [57Sinkunas T. et al.In vitro reconstitution of Cascade-mediated CRISPR immunity in Streptococcus thermophilus.EMBO J. 2013; 32: 385-394Crossref PubMed Scopus (181) Google Scholar]. These nucleases have proven effective at curing plasmids from a bacterial population, thereby eliminating gene expression, and also preventing accidental release of a plasmid into the environment [55Caliando B.J. Voigt C.a Targeted DNA degradation using a CRISPR device stably carried in the host genome.Nat. Commun. 2015; 6: 6989Crossref PubMed Scopus (90) Google Scholar]. CRISPR-Cas9 gene deletion and gene introduction strategies have been demonstrated in an increasing number of industrial microbes since initial proofs of concept were established in 2013 [5Jiang W. et al.RNA-guided editing of bacterial genomes using CRISPR-Cas systems.Nat. Biotechnol. 2013; 31: 233-239Crossref PubMed Scopus (1680) Google Scholar, 56Bikard, D. et al. (2012) CRISPR interference can prevent natural transformation and virulence acquisition during in vivo bacterial infection. 12, 177–186.Google Scholar]. Cellular repair of CRISPR-Cas9-mediated DSBs through the NHEJ pathway can result in the introduction of frame shifts in open reading frames, resulting in a gene knockout (KO) (Figure 3, Key Figure). The programmable KO capabilities afforded by CRISPR-Cas9 can be used to redirect metabolic flux by eliminating genes contributing to competing reactions, such as those involved in the undesirable catabolism of carbon or precursor metabolites. Single-step transformation of multiple single guide RNAs (sgRNAs) is an effective method for the KO of several genes simultaneously in both yeast and bacteria [4Horwitz A.A. et al.Efficient multiplexed integration of synergistic alleles and metabolic pathways in yeasts via CRISPR-Cas.Cell Syst. 2015; 1: 1-9Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 5Jiang W. et al.RNA-guided editing of bacterial genomes using CRISPR-Cas systems.Nat. Biotechnol. 2013; 31: 233-239Crossref PubMed Scopus (1680) Google Scholar, 58Jakočinas T. et al.Multiplex metabolic pathway engineering using CRISPR/Cas9 in Saccharomyces cerevisiae.Metab. Eng. 2015; 28: 213-222Crossref PubMed Scopus (309) Google Scholar]. CRISPR-Cas9 KOs in numerous filamentous fungi species have also been demonstrated, a feat not easily accomplished with incumbent genome-editing technologies [41Nødvig C.S. et al.A CRISPR-Cas9 system for genetic engineering of filamentous fungi.PLoS One. 2015; 10: 1-18Crossref Scopus (378) Google Scholar, 59Liu R. et al.Efficient genome editing in filamentous fungus Trichoderma reesei using the CRISPR/Cas9 system.Cell Discov. 2015; 1: 15007Crossref PubMed Scopus (280) Google Scholar, 60Rong Z. et al.Homologous recombination in human embryonic stem cells using CRISPR/Cas9 nickase and a long DNA donor template.Protein Cell. 2014; 5: 258-260Crossref PubMed Scopus (55) Google Scholar]. In NHEJ-deficient bacteria, the CRISPR-Cas9 system has been utilized in combination with a lambda Red recombinase and short nucleic acid donors that span the Cas9-mediated DSB to knock out genes or introduce small local changes while avoiding DSB-induced cell death [5Jiang W. et al.RNA-guided editing of bacterial genomes using CRISPR-Cas systems.Nat. Biotechnol. 2013; 31: 233-239Crossref PubMed Scopus (1680) Google Scholar, 6Jiang Y. et al.Multigene editing in the Escherichia coli genome via the CRISPR-Cas9 system.Appl. Environ. Microbiol. 2015; 81: 2506-2514Crossref PubMed Scopus (651) Google Scholar]. Industrial biotechnology is reliant on methods of foreign gene introduction to engineer new pathways for biosynthesis of products. Although insertion of exogenous DNA in laboratory yeast is a standard procedure owing to efficient recombination machinery, certain yeast strains lack robust homologous recombination machinery or a well-developed toolset (e.g., selection markers, stable plasmids, etc.) that enable routine modifications [8Mans R. et al.CRISPR/Cas9: a molecular Swiss army knife for simultaneous introduction of multiple genetic modifications in Saccharomyces cerevisiae.FEMS Yeast Res. 2015; 15: 1-15Crossref Scopus (292) Google Scholar]. The advantage of using CRISPR-Cas9 over conventional tools is that the introduction of DSBs greatly increases the rates of recombination when used in conjunction with an appropriate DNA donor molecule [61Jacobs J.Z. et al.Implementation of the CRISPR-Cas9 system in fission yeast.Nat. 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However, the use of CRISPR-Cas9 and multiple sgRNAs, together with multiple donor cassettes, enabled the introduction of biosynthetic production pathways in both Saccharomyces cerevisiae [7Ronda C. et al.CrEdit: CRISPR mediated multi-loci gene integration in Saccharomyces cerevisiae.Microb. Cell Fact. 2015; 14: 1-11Crossref PubMed Scopus (116) Google Scholar, 8Mans R. et al.CRISPR/Cas9: a molecular Swiss army knife for simultaneous introduction of multiple genetic modifications in Saccharomyces cerevisiae.FEMS Yeast Res. 2015; 15: 1-15Crossref Scopus (292) Google Scholar] and Kluyveromyces lactis [4Horwitz A.A. et al.Efficient multiplexed integration of synergistic alleles and metabolic pathways in yeasts via CRISPR-Cas.Cell Syst. 2015; 1: 1-9Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar] in a single transformation event. Conventional genetic engineering efforts in filamentous fungi have lagged behind both the advances and successes seen in bacteria and yeast. The engineering of filamentous fungi is hindered by several factors, including the challenge of delivery through the fungal cell wall, multinucleated cells, and a dearth of available promoters and plasmids. CRISPR-Cas9 does not circumvent all of these limitations, but early experiments engineering KOs in Trichoderma reesei [59Liu R. et al.Efficient genome editing in filamentous fungus Trichoderma reesei using the CRISPR/Cas9 system.Cell Discov. 2015; 1: 15007Crossref PubMed Scopus (280) Google Scholar] and multiple Aspergillus species [41Nødvig C.S. et al.A CRISPR-Cas9 system for genetic engineering of filamentous fungi.PLoS One. 2015; 10: 1-18Crossref Scopus (378) Google Scholar] have demonstrated increased success over classic techniques. Historically, introduction of donor sequences into filamentous fungal genomes has proven difficult, and is often accomplished in engineered NHEJ-deficient strains, which require backcrossing after successful recombination to restore the wild-type DNA repair phenotype [63Zhang C. et al.Highly efficient CRISPR mutagenesis by microhomology-mediated end joining in Aspergillus fumigatus.Fungal Genet. Biol. 2016; 86: 47-57Crossref PubMed Scopus (165) Google Scholar]. In wild-type species, CRISPR-Cas9 has enabled the introduction of heterologous sequences into wild-type Neurospora crassa [64Matsu-ura T. et al.Efficient gene editing in Neurospora crassa with CRISPR technology.Fungal Biol. Biotechnol. 2015; 2: 1-7Crossref PubMed Google Scholar], Aspergillus fumigatus [63Zhang C. et al.Highly efficient CRISPR mutagenesis by microhomology-mediated end joining in Aspergillus fumigatus.Fungal Genet. Biol. 2016; 86: 47-57Crossref PubMed Scopus (165) Google Scholar], and Pyricularia oryzae (anamorph Magnaporthe grisea) [65Arazoe T. et al.Tailor-made CRISPR/Cas system for highly efficient targeted gene replacement in the rice blast fungus.Biotechnol. Bioeng. 2015; 112: 2543-2549Crossref PubMed Scopus (132) Google Scholar]. The recent demonstration of Cas9-mediated multiplexed recombination in Penicillium chrysogenum [66Pohl C. et al.CRISPR/Cas9 based genome editing of Penicillium chrysogenum.ACS Synth. Biol. 2016; 5: 754-764Crossref PubMed Scopus (210) Google Scholar] signals the rapid improvement and imp}, number={2}, journal={TRENDS IN BIOTECHNOLOGY}, publisher={Elsevier BV}, author={Donohoue, Paul D. and Barrangou, Rodolphe and May, Andrew P.}, year={2018}, month={Feb}, pages={134–146} }
 @article{stout_klaenhammer_barrangou_2017, title={CRISPR-Cas Technologies and Applications in Food Bacteria}, volume={8}, ISSN={["1941-1421"]}, DOI={10.1146/annurev-food-072816-024723}, abstractNote={ Clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins form adaptive immune systems that occur in many bacteria and most archaea. In addition to protecting bacteria from phages and other invasive mobile genetic elements, CRISPR-Cas molecular machines can be repurposed as tool kits for applications relevant to the food industry. A primary concern of the food industry has long been the proper management of food-related bacteria, with a focus on both enhancing the outcomes of beneficial microorganisms such as starter cultures and probiotics and limiting the presence of detrimental organisms such as pathogens and spoilage microorganisms. This review introduces CRISPR-Cas as a novel set of technologies to manage food bacteria and offers insights into CRISPR-Cas biology. It primarily focuses on the applications of CRISPR-Cas systems and tools in starter cultures and probiotics, encompassing strain-typing, phage resistance, plasmid vaccination, genome editing, and antimicrobial activity. }, number={1}, journal={ANNUAL REVIEW OF FOOD SCIENCE AND TECHNOLOGY, VOL 8}, publisher={Annual Reviews}, author={Stout, Emily and Klaenhammer, Todd and Barrangou, Rodolphe}, year={2017}, pages={413–437} }
 @misc{hidalgo-cantabrana_o'flaherty_barrangou_2017, title={CRISPR-based engineering of next-generation lactic acid bacteria}, volume={37}, ISSN={["1879-0364"]}, DOI={10.1016/j.mib.2017.05.015}, abstractNote={The advent of CRISPR-based technologies has opened new avenues for the development of next-generation food microorganisms and probiotics with enhanced functionalities. Building off two decades of functional genomics studies unraveling the genetic basis for food fermentations and host-probiotic interactions, CRISPR technologies offer a wide range of opportunities to engineer commercially-relevant Lactobacillus and Bifidobacteria. Endogenous CRISPR-Cas systems can be repurposed to enhance gene expression or provide new features to improve host colonization and promote human health. Alternatively, engineered CRISPR-Cas systems can be harnessed to genetically modify probiotics and enhance their therapeutic potential to deliver vaccines or modulate the host immune response.}, journal={CURRENT OPINION IN MICROBIOLOGY}, publisher={Elsevier BV}, author={Hidalgo-Cantabrana, Claudio and O'Flaherty, Sarah and Barrangou, Rodolphe}, year={2017}, month={Jun}, pages={79–87} }
 @article{hidalgo-cantabrana_crawley_sanchez_barrangou_2017, title={Characterization and Exploitation of CRISPR Loci in Bifidobacterium longum}, volume={8}, ISSN={["1664-302X"]}, DOI={10.3389/fmicb.2017.01851}, abstractNote={Diverse CRISPR-Cas systems provide adaptive immunity in many bacteria and most archaea, via a DNA-encoded, RNA-mediated, nucleic-acid targeting mechanism. Over time, CRISPR loci expand via iterative uptake of invasive DNA sequences into the CRISPR array during the adaptation process. These genetic vaccination cards thus provide insights into the exposure of strains to phages and plasmids in space and time, revealing the historical predatory exposure of a strain. These genetic loci thus constitute a unique basis for genotyping of strains, with potential of resolution at the strain-level. Here, we investigate the occurrence and diversity of CRISPR-Cas systems in the genomes of various Bifidobacterium longum strains across three sub-species. Specifically, we analyzed the genomic content of 66 genomes belonging to B. longum subsp. longum, B. longum subsp. infantis and B. longum subsp. suis, and identified 25 strains that carry 29 total CRISPR-Cas systems. We identify various Type I and Type II CRISPR-Cas systems that are widespread in this species, notably I-C, I-E, and II-C. Noteworthy, Type I-C systems showed extended CRISPR arrays, with extensive spacer diversity. We show how these hypervariable loci can be used to gain insights into strain origin, evolution and phylogeny, and can provide discriminatory sequences to distinguish even clonal isolates. By investigating CRISPR spacer sequences, we reveal their origin and implicate phages and prophages as drivers of CRISPR immunity expansion in this species, with redundant targeting of select prophages. Analysis of CRISPR spacer origin also revealed novel PAM sequences. Our results suggest that CRISPR-Cas immune systems are instrumental in mounting diversified viral resistance in B. longum, and show that these sequences are useful for typing across three subspecies.}, journal={FRONTIERS IN MICROBIOLOGY}, publisher={Frontiers Media SA}, author={Hidalgo-Cantabrana, Claudio and Crawley, Alexandra B. and Sanchez, Borja and Barrangou, Rodolphe}, year={2017}, month={Sep} }
 @article{selle_goh_johnson_sarah_andersen_barrangou_klaenhammer_2017, title={Deletion of Lipoteichoic Acid Synthase Impacts Expression of Genes Encoding Cell Surface Proteins in Lactobacillus acidophilus}, volume={8}, DOI={10.3389/fmicb.2017.00553}, abstractNote={Lactobacillus acidophilus NCFM is a well-characterized probiotic microorganism, supported by a decade of genomic and functional phenotypic investigations. L. acidophilus deficient in lipoteichoic acid (LTA), a major immunostimulant in Gram-positive bacteria, has been shown to shift immune system responses in animal disease models. However, the pleiotropic effects of removing LTA from the cell surface in lactobacilli are unknown. In this study, we surveyed the global transcriptional and extracellular protein profiles of two strains of L. acidophilus deficient in LTA. Twenty-four differentially expressed genes specific to the LTA-deficient strains were identified, including a predicted heavy metal resistance operon and several putative peptidoglycan hydrolases. Cell morphology and manganese sensitivity phenotypes were assessed in relation to the putative functions of differentially expressed genes. LTA-deficient L. acidophilus exhibited elongated cellular morphology and their growth was severely inhibited by elevated manganese concentrations. Exoproteomic surveys revealed distinct changes in the composition and relative abundances of several extracellular proteins and showed a bias of intracellular proteins in LTA-deficient strains of L. acidophilus. Taken together, these results elucidate the impact of ltaS deletion on the transcriptome and extracellular proteins of L. acidophilus, suggesting roles of LTA in cell morphology and ion homeostasis as a structural component of the Gram positive cell wall.}, journal={Frontiers in Microbiology}, publisher={Frontiers Media SA}, author={Selle, Kurt and Goh, Yong J. and Johnson, Brant R. and Sarah, O’Flaherty and Andersen, Joakim M. and Barrangou, Rodolphe and Klaenhammer, Todd R.}, year={2017}, month={Apr} }
 @article{barrangou_gersbach_2017, title={Expanding the CRISPR Toolbox: Targeting RNA with Cas13b}, volume={65}, ISSN={["1097-4164"]}, DOI={10.1016/j.molcel.2017.02.002}, abstractNote={In this issue of Molecular Cell, Smargon et al., 2017Smargon A. Cox D.B.T. Pyzocha N.K. Zheng K. Slaymaker I.M. Gootenberg J.S. Abudayyeh O.A. Essletzbichler P. Shmakov S. Makarova K.S. et al.Mol. Cell. 2017; 65 (this issue): 618-630Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar unearth Cas13b from type VI-B CRISPR-Cas immune systems and characterize its RNA-guided, RNA-targeting activity, including regulation by the novel co-factors Csx27 and Csx28, as well as non-specific collateral RNA damage. In this issue of Molecular Cell, Smargon et al., 2017Smargon A. Cox D.B.T. Pyzocha N.K. Zheng K. Slaymaker I.M. Gootenberg J.S. Abudayyeh O.A. Essletzbichler P. Shmakov S. Makarova K.S. et al.Mol. Cell. 2017; 65 (this issue): 618-630Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar unearth Cas13b from type VI-B CRISPR-Cas immune systems and characterize its RNA-guided, RNA-targeting activity, including regulation by the novel co-factors Csx27 and Csx28, as well as non-specific collateral RNA damage. CRISPR and associated sequences (Cas) together constitute CRISPR-Cas systems that provide adaptive immunity against invasive nucleic acids via DNA-encoded, RNA-mediated, sequence-specific targeting. Several molecular machines derived from CRISPR-Cas systems have been very successfully repurposed as technologies for editing the genome, controlling the transcriptome, and altering the epigenome (Barrangou and Doudna, 2016Barrangou R. Doudna J.A. Nat. Biotechnol. 2016; 34: 933-941Crossref PubMed Scopus (537) Google Scholar). Indeed, Cas9 has enabled the democratization of genome editing in the past 3 years, with great promise for versatile cell engineering. Despite the transformative impact of CRISPR-based technologies in recent years, only a small fraction of CRISPR-Cas systems have yet to be explored in any detail. Consequently, several recent efforts have focused on mining microbial genomes to unearth programmable Cas effector proteins able to expand the molecular biology toolkit, with recent additions such as Cpf1 (now Cas12a) (Zetsche et al., 2015Zetsche B. Gootenberg J.S. Abudayyeh O.O. Slaymaker I.M. Makarova K.S. Essletzbichler P. Volz S.E. Joung J. van der Oost J. Regev A. et al.Cell. 2015; 163: 759-771Abstract Full Text Full Text PDF PubMed Scopus (2500) Google Scholar), C2c2 (now Cas13a) (Shmakov et al., 2015Shmakov S. Abudayyeh O.O. Makarova K.S. Wolf Y.I. Gootenberg J.S. Semenova E. Minakhin L. Joung J. Konermann S. Severinov K. et al.Mol. Cell. 2015; 60: 385-397Abstract Full Text Full Text PDF PubMed Scopus (714) Google Scholar), CasX, and CasY (Burstein et al., 2016Burstein D. Harrington L.B. Strutt S.C. Probst A.J. Anantharaman K. Thomas B.C. Doudna J.A. Banfield J.F. Nature. 2016; (Published online December 22, 2016)https://doi.org/10.1038/nature21059Crossref Scopus (323) Google Scholar). The featured study by Smargon et al., 2017Smargon A. Cox D.B.T. Pyzocha N.K. Zheng K. Slaymaker I.M. Gootenberg J.S. Abudayyeh O.A. Essletzbichler P. Shmakov S. Makarova K.S. et al.Mol. Cell. 2017; 65 (this issue): 618-630Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar reports that Cas13b (previously C2c6) from type VI-B CRISPR-Cas systems is an RNA-guided RNase with an idiosyncratic mechanism of action that could be repurposed to target RNA in a programmable manner (Figure 1). In the past decade, CRISPR-Cas systems have been established as the drivers of adaptive immunity in bacteria and repurposed as a revolutionary genome-editing technology (Barrangou and Doudna, 2016Barrangou R. Doudna J.A. Nat. Biotechnol. 2016; 34: 933-941Crossref PubMed Scopus (537) Google Scholar). Our appreciation for the natural diversity of CRISPR-Cas systems has yielded several rounds of classification and nomenclature evolution, and there are currently two major classes distinguished by single versus complexed effector proteins, which are further divided into six major types and 19 subtypes (Makarova et al., 2015Makarova K.S. Wolf Y.I. Alkhnbashi O.S. Costa F. Shah S.A. Saunders S.J. Barrangou R. Brouns S.J. Charpentier E. Haft D.H. et al.Nat. Rev. Microbiol. 2015; 13: 722-736Crossref PubMed Scopus (1462) Google Scholar, Shmakov et al., 2017Shmakov S. Smargon A. Scott D. Cox D. Pyzocha N. Yan W. Abudayyeh O.O. Gootenberg J.S. Makarova K.S. Wolf Y.I. et al.Nat. Rev. Microbiol. 2017; (Published online January 23, 2017)https://doi.org/10.1038/nrmicro.2016.184Crossref PubMed Scopus (555) Google Scholar). The most effective strategy to uncover the next Cas effector protein is to develop sophisticated and creative in silico analyses to parse through the ever-increasing numbers of sequenced microbial genomes. To find a new subtype of CRISPR-Cas systems, Smargon et al., 2017Smargon A. Cox D.B.T. Pyzocha N.K. Zheng K. Slaymaker I.M. Gootenberg J.S. Abudayyeh O.A. Essletzbichler P. Shmakov S. Makarova K.S. et al.Mol. Cell. 2017; 65 (this issue): 618-630Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar used a computational database mining approach to search for single large effector proteins associated with CRISPR arrays that did not contain the nearly universal markers cas1 and cas2 and identified Cas13b. Two distinct classes were detected repeatedly among a total of 105 genomic loci identified and were particularly enriched in Bacteriodetes, such as Porphyromonas and Prevotella. Though Cas13b has a novel sequence, it carries two HEPN domains (RxxxxH), somewhat similar to the architecture of Cas13a, the signature RNase from the type VI-A CRISPR-Cas system (Shmakov et al., 2015Shmakov S. Abudayyeh O.O. Makarova K.S. Wolf Y.I. Gootenberg J.S. Semenova E. Minakhin L. Joung J. Konermann S. Severinov K. et al.Mol. Cell. 2015; 60: 385-397Abstract Full Text Full Text PDF PubMed Scopus (714) Google Scholar, Shmakov et al., 2017Shmakov S. Smargon A. Scott D. Cox D. Pyzocha N. Yan W. Abudayyeh O.O. Gootenberg J.S. Makarova K.S. Wolf Y.I. et al.Nat. Rev. Microbiol. 2017; (Published online January 23, 2017)https://doi.org/10.1038/nrmicro.2016.184Crossref PubMed Scopus (555) Google Scholar). Genetically, the CRISPR array is reminiscent of other class 2 systems, with a 36 nt, partially palindromic repeat sequence and spacers with homology to phage genome sequences. Mechanistically, two mature CRISPR RNA (crRNA) species were identified, a "short" 66 nt crRNA and a "long" 118 nt crRNA with an extended CRISPR repeat portion. After porting the systems into E. coli, Smargon et al., 2017Smargon A. Cox D.B.T. Pyzocha N.K. Zheng K. Slaymaker I.M. Gootenberg J.S. Abudayyeh O.A. Essletzbichler P. Shmakov S. Makarova K.S. et al.Mol. Cell. 2017; 65 (this issue): 618-630Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar showed knockdown of essential genes when Cas13b was heterologously expressed with targeted crRNAs. By studying strongly depleted sequences, Smargon et al., 2017Smargon A. Cox D.B.T. Pyzocha N.K. Zheng K. Slaymaker I.M. Gootenberg J.S. Abudayyeh O.A. Essletzbichler P. Shmakov S. Makarova K.S. et al.Mol. Cell. 2017; 65 (this issue): 618-630Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar revealed that heavily targeted sequences are typically flanked by a peculiar double-sided protospacer flanking sequence (PFS), akin to the protospacer-associated motif (PAM). Focusing on the Bergeyella zoohelcum BzCas13b, Smargon et al., 2017Smargon A. Cox D.B.T. Pyzocha N.K. Zheng K. Slaymaker I.M. Gootenberg J.S. Abudayyeh O.A. Essletzbichler P. Shmakov S. Makarova K.S. et al.Mol. Cell. 2017; 65 (this issue): 618-630Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar showed sequence-specific single-stranded RNA (ssRNA) targeting. Furthermore, this activity was associated with non-specific RNA cleavage, dubbed "collateral" RNase activity, albeit only in the presence of target RNA, similar to what has been previously described for Cas13a (Abudayyeh et al., 2016Abudayyeh O.O. Gootenberg J.S. Konermann S. Joung J. Slaymaker I.M. Cox D.B. Shmakov S. Makarova K.S. Semenova E. Minakhin L. et al.Science. 2016; 353: aaf5573Crossref PubMed Scopus (1146) Google Scholar, East-Seletsky et al., 2016East-Seletsky A. O'Connell M.R. Knight S.C. Burstein D. Cate J.H. Tjian R. Doudna J.A. Nature. 2016; 538: 270-273Crossref PubMed Scopus (592) Google Scholar). HEPN-dependent interference was confirmed by resistance against lytic phage MS2, with reduced plaque formation in the presence of targeting spacers, involving conserved catalytic arginines and histidines (R116/H121 and R1177/H1182). Additionally, the study investigated other novel cas genes they found typically associated with cas13b, Csx27, and Csx28, which were determined to be a repressor and an enhancer of Cas13b activity, respectively. Mechanistically, the absence of the nearly universal CRISPR markers cas1 and cas2 is intriguing given their implication in novel spacer acquisition during the immunization process, though they could be provided in trans given the frequent occurrence of other CRISPR-Cas systems in genomes that carry cas13b. This is somewhat contradictory with the typical orthogonality of different CRISPR-Cas systems, and future studies should investigate whether Cas1 and Cas2 can interact with type VI-B CRISPR arrays and determine how acquisition occurs in this subtype. The potential for cross-reactivity of acquisition machinery across systems may help with understanding the currently mysterious mechanisms of immunization. Arguably the most novel insight provided by this new system is the discovery of the co-regulatory molecules Csx27 and Csx28. The dual control options afforded by Csx27 inhibition and Csx28 activation of Cas13b beg the question as to whether there is control of cas13b transcription prior to interference and/or post-transcriptional control by direct interaction with Cas13b (or possibly crRNA) following invasive RNA targeting. Smargon et al., 2017Smargon A. Cox D.B.T. Pyzocha N.K. Zheng K. Slaymaker I.M. Gootenberg J.S. Abudayyeh O.A. Essletzbichler P. Shmakov S. Makarova K.S. et al.Mol. Cell. 2017; 65 (this issue): 618-630Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar's demonstration of Csx27- and Csx28-based regulation in non-native hosts from heterologous systems indicates that the latter is at least one contributing factor, but this should certainly be a necessary area of future work. The mechanism of Csx27 and Csx28 action also determines how they might interplay with the putative target phages, including potential roles in the rise of phage resistance via invader transcript destruction or possibly triggering of host death via collateral RNA targeting. The former would enable the host to thrive, whereas the latter would drive suicide of infected cells for the benefit of the rest of the population, akin to the abortive infection system. It will be important to define the role of these new regulatory control systems in determining the cellular outcome of Cas13b activity and the impact at the population levels for both hosts and phages. Additionally, similar to recent analyses of Cas9 and other Cas effector proteins, biochemical and structural studies will also be necessary, to provide critical insights into their biology, as well as a basis to enhance their functions and optimize their activity and specificity by engineering. Given the potential repurposing of these molecular machines in eukaryotes, it is intriguing to ponder how effectively and specifically RNA targeting will be for either RNA-virus eradication or promoting programmed cell death by exploiting collateral RNase activity. Actually, it is still unclear whether this system naturally targets RNA viruses and/or the RNA transcripts of DNA viruses. Although CRISPR-Cas systems are potent antivirals by nature, they could also be reprogrammed to drive the death of the host when it is desirable to do so. Indeed, an endogenous lethal self-targeting pathway could be hijacked to drive cell suicide by exploiting the collateral RNA damage mechanism, leading to systemic RNA degradation and programmed cell death. This is reminiscent of the repurposing of self-targeting CRISPR-Cas systems as antimicrobials (Gomaa et al., 2014Gomaa A.A. Klumpe H.E. Luo M.L. Selle K. Barrangou R. Beisel C.L. MBio. 2014; 5: e00928-13Crossref PubMed Scopus (245) Google Scholar). Overall, this study illustrates how mining dark matter in obscure bacterial genomes is continuing to yield novel Cas-based molecular machines that advance our understanding of the interplay between bacteria and their predators and open new avenues for the development of new tools that expand the molecular biology toolbox for genome, transcriptome, and epigenome engineering. R.B. and C.A.G. are inventors on patents related to CRISPR-Cas systems and their various uses. R.B. is a co-founder and SAB member of Intellia Therapeutics and Locus Biosciences; C.A.G. is a co-founder and SAB member of Locus Biosciences and Element Genomics. Cas13b Is a Type VI-B CRISPR-Associated RNA-Guided RNase Differentially Regulated by Accessory Proteins Csx27 and Csx28Smargon et al.Molecular CellJanuary 5, 2017In BriefSmargon et al. identify and characterize two class 2 type VI-B CRISPR systems lacking Cas1 and Cas2 and containing the RNA-guided RNase Cas13b, differentially regulated by Csx27 and Csx28. Through an E. coli essential gene screen they show that Cas13b RNA targeting is dependent on a double-sided PFS and RNA accessibility. Full-Text PDF Open Archive}, number={4}, journal={MOLECULAR CELL}, publisher={Elsevier BV}, author={Barrangou, Rodolphe and Gersbach, Charles A.}, year={2017}, month={Feb}, pages={582–584} }
 @article{pijkeren_barrangou_2017, title={Genome Editing of Food-Grade Lactobacilli To Develop Therapeutic Probiotics}, volume={5}, DOI={10.1128/microbiolspec.bad-0013-2016}, abstractNote={ABSTRACTLactic acid bacteria have been used historically for food manufacturing mainly to ensure preservation via fermentation. More recently, lactic acid bacteria have been exploited to promote human health, and many strains serve as industrial workhorses. Recent advances in microbiology and molecular biology have contributed to understanding the genetic basis of many of their functional attributes. These include dissection of biochemical processes that drive food fermentation, and identification and characterization of health-promoting features that positively impact the composition and roles of microbiomes in human health. Recently, the advent of clustered regularly interspaced short palindromic repeat (CRISPR)-based technologies has revolutionized our ability to manipulate genomes, and we are on the cusp of a broad-scale genome editing revolution. Here, we discuss recent advances in genetic alteration of food-grade bacteria, with a focus on CRISPR-associated enzyme genome editing, single-stranded DNA recombineering, and the modification of bacteriophages. These tools open new avenues for the genesis of next-generation biotherapeutic agents with improved genotypes and enhanced health-promoting functional features.}, number={5}, journal={Microbiology Spectrum}, publisher={American Society for Microbiology}, author={Pijkeren, Jan-Peter and Barrangou, Rodolphe}, year={2017}, month={Oct} }
 @article{barrangou_bikard_2017, title={Guest editorial: CRISPRcas9: CRISPR-Cas systems: at the cutting edge of microbiology}, volume={37}, DOI={10.1016/j.mib.2017.09.015}, journal={Current Opinion in Microbiology}, publisher={Elsevier BV}, author={Barrangou, Rodolphe and Bikard, David}, year={2017}, month={Jun}, pages={vii-viii} }
 @article{weissman_holmes_barrangou_moineau_fagan_levin_johnson_2017, title={Immune Loss as a Driver of Coexistence During Host-Phage Coevolution}, url={https://doi.org/10.1101/105908}, DOI={10.1101/105908}, abstractNote={AbstractBacteria and their viral pathogens face constant pressure for augmented immune and infective capabilities, respectively. Under this reciprocally imposed selective regime, we expect to see a runaway evolutionary arms race, ultimately leading to the extinction of one species. Despite this prediction, in many systems host and pathogen coexist with minimal coevolution even when well-mixed. Previous work explained this puzzling phenomenon by invoking fitness tradeoffs, which can diminish an arms race dynamic. Here we propose that the regular loss of immunity by the bacterial host can also produce host-phage coexistence. We pair a general model of immunity with an experimental and theoretical case study of the CRISPR-Cas immune system to contrast the behavior of tradeoff and loss mechanisms in well-mixed systems. We find that, while both mechanisms can produce stable coexistence, only immune loss does so robustly within realistic parameter ranges.}, author={Weissman, Jake L and Holmes, Rayshawn and Barrangou, Rodolphe and Moineau, Sylvain and Fagan, William F and Levin, Bruce and Johnson, Philip L F}, year={2017}, month={Feb} }
 @article{klotz_o'flaherty_goh_barrangou_2017, title={Investigating the Effect of Growth Phase on the Surface-Layer Associated Proteome of Lactobacillus acidophilus Using Quantitative Proteomics}, volume={8}, ISSN={["1664-302X"]}, DOI={10.3389/fmicb.2017.02174}, abstractNote={Bacterial surface-layers (S-layers) are semi-porous crystalline arrays that self-assemble to form the outermost layer of some cell envelopes. S-layers have been shown to act as scaffolding structures for the display of auxiliary proteins externally. These S-layer associated proteins have recently gained attention in probiotics due to their direct physical contact with the intestinal mucosa and potential role in cell proliferation, adhesion, and immunomodulation. A number of studies have attempted to catalog the S-layer associated proteome of Lactobacillus acidophilus NCFM under a single condition. However, due to the versatility of the cell surface, we chose to employ a multiplexing-based approach with the intention of accurately contrasting multiple conditions. In this study, a previously described lithium chloride isolation protocol was used to release proteins bound to the L. acidophilus S-layer during logarithmic and early stationary growth phases. Protein quantification values were obtained via TMT (tandem mass tag) labeling combined with a triple-stage mass spectrometry (MS3) method. Results showed significant growth stage-dependent alterations to the surface-associated proteome while simultaneously highlighting the sensitivity and reproducibility of the technology. Thus, this study establishes a framework for quantifying condition-dependent changes to cell surface proteins that can easily be applied to other S-layer forming bacteria.}, journal={FRONTIERS IN MICROBIOLOGY}, publisher={Frontiers Media SA}, author={Klotz, Courtney and O'Flaherty, Sarah and Goh, Yong Jun and Barrangou, Rodolphe}, year={2017}, month={Nov} }
 @article{theilmann_goh_nielsen_klaenhammer_barrangou_abou hachem_2017, title={Lactobacillus acidophilus Metabolizes Dietary Plant Glucosides and Externalizes Their Bioactive Phytochemicals}, volume={8}, ISSN={["2150-7511"]}, url={https://doi.org/10.1128/mBio.01421-17}, DOI={10.1128/mbio.01421-17}, abstractNote={ABSTRACT
          
            Therapeutically active glycosylated phytochemicals are ubiquitous in the human diet. The human gut microbiota (HGM) modulates the bioactivities of these compounds, which consequently affect host physiology and microbiota composition. Despite a significant impact on human health, the key players and the underpinning mechanisms of this interplay remain uncharacterized. Here, we demonstrate the growth of
            Lactobacillus acidophilus
            on mono- and diglucosyl dietary plant glycosides (PGs) possessing small aromatic aglycones. Transcriptional analysis revealed the upregulation of host interaction genes and identified two loci that encode phosphotransferase system (PTS) transporters and phospho-β-glucosidases, which mediate the uptake and deglucosylation of these compounds, respectively. Inactivating these transport and hydrolysis genes abolished or severely reduced growth on PG, establishing the specificity of the loci to distinct groups of PGs. Following intracellular deglucosylation, the aglycones of PGs are externalized, rendering them available for absorption by the host or for further modification by other microbiota taxa. The PG utilization loci are conserved in
            L. acidophilus
            and closely related lactobacilli, in correlation with versatile growth on these compounds. Growth on the tested PG appeared more common among human gut lactobacilli than among counterparts from other ecologic niches. The PGs that supported the growth of
            L. acidophilus
            were utilized poorly or not at all by other common HGM strains, underscoring the metabolic specialization of
            L. acidophilus
            . These findings highlight the role of human gut
            L. acidophilus
            and select lactobacilli in the bioconversion of glycoconjugated phytochemicals, which is likely to have an important impact on the HGM and human host.
          
          
            IMPORTANCE
            Thousands of therapeutically active plant-derived compounds are widely present in berries, fruits, nuts, and beverages like tea and wine. The bioactivity and bioavailability of these compounds, which are typically glycosylated, are altered by microbial bioconversions in the human gut. Remarkably, little is known about the bioconversion of PGs by the gut microbial community, despite the significance of this metabolic facet to human health. Our work provides the first molecular insights into the metabolic routes of diet relevant and therapeutically active PGs by
            Lactobacillus acidophilus
            and related human gut lactobacilli. This taxonomic group is adept at metabolizing the glucoside moieties of select PG and externalizes their aglycones. The study highlights an important role of lactobacilli in the bioconversion of dietary PG and presents a framework from which to derive molecular insights into their metabolism by members of the human gut microbiota.
          }, number={6}, journal={MBIO}, publisher={American Society for Microbiology}, author={Theilmann, Mia C. and Goh, Yong Jun and Nielsen, Kristian Fog and Klaenhammer, Todd R. and Barrangou, Rodolphe and Abou Hachem, Maher}, editor={Martens, Eric and McFall-Ngai, Margaret J.Editors}, year={2017} }
 @article{bikard_barrangou_2017, title={Les systèmes CRISPR-Cas comme arme contre les bactéries pathogènes}, volume={211}, ISSN={2105-0678 2105-0686}, url={http://dx.doi.org/10.1051/JBIO/2018004}, DOI={10.1051/jbio/2018004}, abstractNote={CRISPR-Cas systems (Clustered Regularly Interspaced Short Palindromic Repeats) are the adaptive immune system of bacteria and archaea. They target foreign genetic elements thanks to small RNAs able to guide Cas nucleases to destroy them. These nucleases can be reprogrammed to target chromosomal sequences rather than invasive genetic elements. Whereas targeting the genome of eukaryotic cells enables the efficient genesis of mutations, DNA breaks induced by Cas nucleases are lethal in bacteria. This property can be used in the development of novel antimicrobial strategies. CRISPR-Cas systems can be delivered to target bacteria using bacteriophage capsids in order to specifically eliminate bacteria carrying antibiotic resistance genes or virulence factors. These technologies enable the development of novel tools based on CRISPR-Cas systems to specifically eliminate pathogenic bacteria and precisely modify the composition of various microbiomes.}, number={4}, journal={Biologie Aujourd'hui}, publisher={EDP Sciences}, author={Bikard, David and Barrangou, Rodolphe}, year={2017}, pages={265–270} }
 @article{toms_barrangou_2017, title={On the global CRISPR array behavior in class I systems}, volume={12}, ISSN={["1745-6150"]}, DOI={10.1186/s13062-017-0193-2}, abstractNote={Much effort is underway to build and upgrade databases and tools related to occurrence, diversity, and characterization of CRISPR-Cas systems. As microbial communities and their genome complements are unearthed, much emphasis has been placed on details of individual strains and model systems within the CRISPR-Cas classification, and that collection of information as a whole affords the opportunity to analyze CRISPR-Cas systems from a quantitative perspective to gain insight into distribution of CRISPR array sizes across the different classes, types and subtypes. CRISPR diversity, nomenclature, occurrence, and biological functions have generated a plethora of data that created a need to understand the size and distribution of these various systems to appreciate their features and complexity.By utilizing a statistical framework and visual analytic techniques, we have been able to test several hypotheses about CRISPR loci in bacterial class I systems. Quantitatively, though CRISPR loci can expand to hundreds of spacers, the mean and median sizes are 40 and 25, respectively, reflecting rather modest acquisition and/or retention overall. Histograms uncovered that CRISPR array size displayed a parametric distribution, which was confirmed by a goodness-of fit test. Mapping the frequency of CRISPR loci on a standardized chromosome plot revealed that CRISPRs have a higher probability of occurring at clustered locations along the positive or negative strand. Lastly, when multiple arrays occur in a particular system, the size of a particular CRISPR array varies with its distance from the cas operon, reflecting acquisition and expansion biases.This study establishes that bacterial Class I CRISPR array size tends to follow a geometric distribution; these CRISPRs are not randomly distributed along the chromosome; and the CRISPR array closest to the cas genes is typically larger than loci in trans. Overall, we provide an analytical framework to understand the features and behavior of CRISPR-Cas systems through a quantitative lens.This article was reviewed by Eugene Koonin (NIH-NCBI) and Uri Gophna (Tel Aviv University).}, journal={BIOLOGY DIRECT}, author={Toms, Alice and Barrangou, Rodolphe}, year={2017}, month={Aug} }
 @article{nair_2017, title={QnAs with Rodolphe Barrangou}, volume={114}, DOI={10.1073/pnas.1710348114}, abstractNote={The past decade in biological research might well be christened the age of CRISPR, a once-curious feature of bacterial genomes that spawned a handy tool for editing genes. Using CRISPR-based tools, researchers are making leaps in basic clinical research, and biotechnology companies are racing to launch trials of gene therapies for an array of diseases. Yet the immediate gains from this game-changing technique might spring from its application to agriculture. Hornless dairy cattle, drought-resistant wheat, and nonbrowning mushrooms are merely the harbingers of an approaching agricultural revolution, says Rodolphe Barrangou, a molecular biologist and food scientist at North Carolina State University. Barrangou’s foresight stems from his long familiarity with CRISPR. More than a decade ago, while working at the Danish food ingredient manufacturer Danisco (now DuPont), Barrangou furnished experimental proof for the notion that CRISPR confers a form of adaptive immunity that helps bacteria fend off invading viruses. For this crucial insight into the fundamental biology of CRISPR, Barrangou was honored with 2017 National Academy of Sciences Award in molecular biology. PNAS spoke to Barrangou about his wide-ranging work on CRISPR.



Rodolphe Barrangou. Image courtesy of North Carolina State University (Raleigh, NC).



> PNAS:CRISPR entered the spotlight when its potential as a genome editor became apparent, but your tryst with it began more than a decade ago while working with Philippe Horvath in the food industry. Those efforts led to a milestone 2007 article in Science , in which you demonstrated that bacteria use CRISPR-Cas systems as a form of adaptive immunity against viruses (1). Could you take our readers down memory lane?

> Barrangou:For a long time, people didn’t really have a clue what these repeated DNA sequences—the CRISPR arrays—in …}, number={28}, journal={Proceedings of the National Academy of Sciences}, publisher={Proceedings of the National Academy of Sciences}, author={Nair, Prashant}, year={2017}, month={Jul}, pages={7183–7184} }
 @article{barrangou_ousterout_2017, title={Repurposing CRISPR-Cas systems as DNA-based smart antimicrobials}, volume={3}, DOI={10.18609/cgti.2017.008}, number={1}, journal={Cell and Gene Therapy Insights}, publisher={BioInsights Publishing, Ltd.}, author={Barrangou, Rodolphe and Ousterout, David G}, year={2017}, month={Feb}, pages={63–72} }
 @article{johnson_o'flaherty_goh_carroll_barrangou_klaenhammer_2017, title={The S-layer Associated Serine Protease Homolog PrtX Impacts Cell Surface-Mediated Microbe-Host Interactions of Lactobacillus acidophilus NCFM}, volume={8}, ISSN={["1664-302X"]}, DOI={10.3389/fmicb.2017.01185}, abstractNote={Health-promoting aspects attributed to probiotic microorganisms, including adhesion to intestinal epithelia and modulation of the host mucosal immune system, are mediated by proteins found on the bacterial cell surface. Notably, certain probiotic and commensal bacteria contain a surface (S-) layer as the outermost stratum of the cell wall. S-layers are non-covalently bound semi-porous, crystalline arrays of self-assembling, proteinaceous subunits called S-layer proteins (SLPs). Recent evidence has shown that multiple proteins are non-covalently co-localized within the S-layer, designated S-layer associated proteins (SLAPs). In Lactobacillus acidophilus NCFM, SLP and SLAPs have been implicated in both mucosal immunomodulation and adhesion to the host intestinal epithelium. In this study, a S-layer associated serine protease homolog, PrtX (prtX, lba1578), was deleted from the chromosome of L. acidophilus NCFM. Compared to the parent strain, the PrtX-deficient strain (ΔprtX) demonstrated increased autoaggregation, an altered cellular morphology, and pleiotropic increases in adhesion to mucin and fibronectin, in vitro. Furthermore, ΔprtX demonstrated increased in vitro immune stimulation of IL-6, IL-12, and IL-10 compared to wild-type, when exposed to mouse dendritic cells. Finally, in vivo colonization of germ-free mice with ΔprtX led to an increase in epithelial barrier integrity. The absence of PrtX within the exoproteome of a ΔprtX strain caused morphological changes, resulting in a pleiotropic increase of the organisms’ immunomodulatory properties and interactions with some intestinal epithelial cell components.}, journal={FRONTIERS IN MICROBIOLOGY}, publisher={Frontiers Media SA}, author={Johnson, Brant R. and O'Flaherty, Sarah and Goh, Yong Jun and Carroll, Ian and Barrangou, Rodolphe and Klaenhammer, Todd R.}, year={2017}, month={Jun} }
 @misc{bikard_barrangou_2017, title={Using CRISPR-Cas systems as antimicrobials}, volume={37}, ISSN={["1879-0364"]}, DOI={10.1016/j.mib.2017.08.005}, abstractNote={Although CRISPR-Cas systems naturally evolved to provide adaptive immunity in bacteria and archaea, Cas nucleases can be co-opted to target chromosomal sequences rather than invasive genetic elements. Although genome editing is the primary outcome of self-targeting using CRISPR-based technologies in eukaryotes, self-targeting by CRISPR is typically lethal in bacteria. Here, we discuss how DNA damage introduced by Cas nucleases in bacteria can efficiently and specifically lead to plasmid curing or drive cell death. Specifically, we discuss how various CRISPR-Cas systems can be engineered and delivered using phages or phagemids as vectors. These principles establish CRISPR-Cas systems as potent and programmable antimicrobials, and open new avenues for the development of CRISPR-based tools for selective removal of bacterial pathogens and precise microbiome composition alteration.}, journal={CURRENT OPINION IN MICROBIOLOGY}, publisher={Elsevier BV}, author={Bikard, David and Barrangou, Rodolphe}, year={2017}, month={Jun}, pages={155–160} }
 @misc{barrangou_doudna_2016, title={Applications of CRISPR technologies in research and beyond}, volume={34}, ISSN={["1546-1696"]}, url={https://doi.org/10.1038/nbt.3659}, DOI={10.1038/nbt.3659}, abstractNote={Programmable DNA cleavage using CRISPR-Cas9 enables efficient, site-specific genome engineering in single cells and whole organisms. In the research arena, versatile CRISPR-enabled genome editing has been used in various ways, such as controlling transcription, modifying epigenomes, conducting genome-wide screens and imaging chromosomes. CRISPR systems are already being used to alleviate genetic disorders in animals and are likely to be employed soon in the clinic to treat human diseases of the eye and blood. Two clinical trials using CRISPR-Cas9 for targeted cancer therapies have been approved in China and the United States. Beyond biomedical applications, these tools are now being used to expedite crop and livestock breeding, engineer new antimicrobials and control disease-carrying insects with gene drives.}, number={9}, journal={NATURE BIOTECHNOLOGY}, publisher={Springer Nature}, author={Barrangou, Rodolphe and Doudna, Jennifer A.}, year={2016}, month={Sep}, pages={933–941} }
 @article{andersen_shoup_robinson_bitton_olsen_barrangou_2016, title={CRISPR Diversity and Microevolution in Clostridium difficile}, volume={8}, ISSN={["1759-6653"]}, DOI={10.1093/gbe/evw203}, abstractNote={Abstract Virulent strains of Clostridium difficile have become a global health problem associated with morbidity and mortality. Traditional typing methods do not provide ideal resolution to track outbreak strains, ascertain genetic diversity between isolates, or monitor the phylogeny of this species on a global basis. Here, we investigate the occurrence and diversity of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated genes (cas) in C. difficile to assess the potential of CRISPR-based phylogeny and high-resolution genotyping. A single Type-IB CRISPR-Cas system was identified in 217 analyzed genomes with cas gene clusters present at conserved chromosomal locations, suggesting vertical evolution of the system, assessing a total of 1,865 CRISPR arrays. The CRISPR arrays, markedly enriched (8.5 arrays/genome) compared with other species, occur both at conserved and variable locations across strains, and thus provide a basis for typing based on locus occurrence and spacer polymorphism. Clustering of strains by array composition correlated with sequence type (ST) analysis. Spacer content and polymorphism within conserved CRISPR arrays revealed phylogenetic relationship across clades and within ST. Spacer polymorphisms of conserved arrays were instrumental for differentiating closely related strains, e.g., ST1/RT027/B1 strains and pathogenicity locus encoding ST3/RT001 strains. CRISPR spacers showed sequence similarity to phage sequences, which is consistent with the native role of CRISPR-Cas as adaptive immune systems in bacteria. Overall, CRISPR-Cas sequences constitute a valuable basis for genotyping of C. difficile isolates, provide insights into the micro-evolutionary events that occur between closely related strains, and reflect the evolutionary trajectory of these genomes.}, number={9}, journal={GENOME BIOLOGY AND EVOLUTION}, publisher={Oxford University Press (OUP)}, author={Andersen, Joakim M. and Shoup, Madelyn and Robinson, Cathy and Bitton, Robert and Olsen, Katharina E. P. and Barrangou, Rodolphe}, year={2016}, month={Sep}, pages={2841–2855} }
 @article{barrangou_dudley_2016, title={CRISPR-Based Typing and Next-Generation Tracking Technologies}, volume={7}, DOI={10.1146/annurev-food-022814-015729}, abstractNote={ Bacteria occur ubiquitously in nature and are broadly relevant throughout the food supply chain, with diverse and variable tolerance levels depending on their origin, biological role, and impact on the quality and safety of the product as well as on the health of the consumer. With increasing knowledge of and accessibility to the microbial composition of our environments, food supply, and host-associated microbiota, our understanding of and appreciation for the ratio of beneficial to undesirable bacteria are rapidly evolving. Therefore, there is a need for tools and technologies that allow definite, accurate, and high-resolution identification and typing of various groups of bacteria that include beneficial microbes such as starter cultures and probiotics, innocuous commensals, and undesirable pathogens and spoilage organisms. During the transition from the current molecular biology–based PFGE (pulsed-field gel electrophoresis) gold standard to the increasingly accessible omics-level whole-genome sequencing (WGS) N-gen standard, high-resolution technologies such as CRISPR-based genotyping constitute practical and powerful alternatives that provide valuable insights into genome microevolution and evolutionary trajectories. Indeed, several studies have shown potential for CRISPR-based typing of industrial starter cultures, health-promoting probiotic strains, animal commensal species, and problematic pathogens. Emerging CRISPR-based typing methods open new avenues for high-resolution typing of a broad range of bacteria and constitute a practical means for rapid tracking of a diversity of food-associated microbes. }, number={1}, journal={Annual Review of Food Science and Technology}, publisher={Annual Reviews}, author={Barrangou, Rodolphe and Dudley, Edward G.}, year={2016}, month={Feb}, pages={395–411} }
 @misc{barrangou_dudley_2016, title={CRISPR-based typing and next-generation tracking technologies}, volume={7}, journal={Annual review of food science and technology, vol 7}, author={Barrangou, R. and Dudley, E. G.}, year={2016}, pages={395–411} }
 @misc{briner_barrangou_2016, title={Deciphering and shaping bacterial diversity through CRISPR}, volume={31}, ISSN={["1879-0364"]}, url={https://doi.org/10.1016/j.mib.2016.03.006}, DOI={10.1016/j.mib.2016.03.006}, abstractNote={Phage and bacteria have engaged in a sustainable arms race, a seemingly endless conflict, since the beginning of time. CRISPR-Cas systems shape and generate environmental diversity through evolution of both predator and prey genomes. Indeed, the gain or loss of CRISPR-mediated immunity and genome maintenance can spark speciation in bacteria. Alternatively, turning CRISPR-Cas on the host by targeting chromosomal DNA has led to the development of next-generation smart antimicrobials and genetic screening and engineering technologies. Although the ability to target and cleave DNA in a sequence-specific manner is a powerful mechanism utilized by bacteria to fend off phage, plasmids, and potentially harmful nucleic acids, it is also a promising technology for programmable targeting of undesirable bacteria in microbiome consortia.}, journal={CURRENT OPINION IN MICROBIOLOGY}, publisher={Elsevier BV}, author={Briner, Alexandra E. and Barrangou, Rodolphe}, year={2016}, month={Jun}, pages={101–108} }
 @article{hymes_johnson_barrangou_klaenhammer_2016, title={Functional Analysis of an S-Layer-Associated Fibronectin-Binding Protein in Lactobacillus acidophilus NCFM}, volume={82}, ISSN={["1098-5336"]}, DOI={10.1128/aem.00024-16}, abstractNote={ABSTRACT
          
            Bacterial surface layers (S-layers) are crystalline arrays of self-assembling proteinaceous subunits called S-layer proteins (Slps) that comprise the outermost layer of the cell envelope. Many additional proteins that are associated with or embedded within the S-layer have been identified in
            Lactobacillus acidophilus
            NCFM, an S-layer-forming bacterium that is widely used in fermented dairy products and probiotic supplements. One putative S-layer-associated protein (SLAP), LBA0191, was predicted to mediate adhesion to fibronectin based on the
            in silico
            detection of a fibronectin-binding domain. Fibronectin is a major component of the extracellular matrix (ECM) of intestinal epithelial cells. Adhesion to intestinal epithelial cells is considered an important trait for probiotic microorganisms during transit and potential association with the intestinal mucosa. To investigate the functional role of LBA0191 (designated FbpB) in
            L. acidophilus
            NCFM, an
            fbpB
            -deficient strain was constructed. The
            L. acidophilus
            mutant with a deletion of
            fbpB
            lost the ability to adhere to mucin and fibronectin
            in vitro
            . Homologues of
            fbpB
            were identified in five additional putative S-layer-forming species, but no homologues were detected in species outside the
            L. acidophilus
            homology group.
          }, number={9}, journal={APPLIED AND ENVIRONMENTAL MICROBIOLOGY}, publisher={American Society for Microbiology}, author={Hymes, Jeffrey P. and Johnson, Brant R. and Barrangou, Rodolphe and Klaenhammer, Todd R.}, editor={Dudley, E. G.Editor}, year={2016}, month={May}, pages={2676–2685} }
 @article{morovic_hibberd_zabel_barrangou_stahl_2016, title={Genotyping by PCR and High-Throughput Sequencing of Commercial Probiotic Products Reveals Composition Biases}, volume={7}, ISSN={["1664-302X"]}, DOI={10.3389/fmicb.2016.01747}, abstractNote={Recent advances in microbiome research have brought renewed focus on beneficial bacteria, many of which are available in food and dietary supplements. Although probiotics have historically been defined as microorganisms that convey health benefits when ingested in sufficient viable amounts, this description now includes the stipulation “well defined strains,” encompassing definitive taxonomy for consumer consideration and regulatory oversight. Here, we evaluated 52 commercial dietary supplements covering a range of labeled species using plate counting and targeted genotyping. Strain identities were assessed using methods recently published by the United States Pharmacopeial Convention. We also determined the relative abundance of individual bacteria by high-throughput sequencing (HTS) of the 16S rRNA sequence using paired-end 2 × 250 bp Illumina MiSeq technology. Using these methods, we tested the hypothesis that products do contain the quantitative and qualitative list of labeled microbial species. We found that 17 samples (33%) were below label claim for CFU prior to their expiration dates. A multiplexed-PCR scheme showed that only 30/52 (58%) of the products contained a correctly labeled classification, with issues encompassing incorrect taxonomy, missing species, and un-labeled species. The HTS revealed that many blended products consisted predominantly of Lactobacillus acidophilus and Bifidobacterium animalis subsp. lactis. These results highlight the need for reliable methods to determine the correct taxonomy and quantify the relative amounts of mixed microbial populations in commercial probiotic products.}, journal={FRONTIERS IN MICROBIOLOGY}, publisher={Frontiers Media SA}, author={Morovic, Wesley and Hibberd, Ashley A. and Zabel, Bryan and Barrangou, Rodolphe and Stahl, Buffy}, year={2016}, month={Nov} }
 @article{briner_barrangou_2016, title={Guide RNAs: A Glimpse at the Sequences that Drive CRISPR–Cas Systems}, volume={2016}, DOI={10.1101/pdb.top090902}, abstractNote={CRISPR–Cas systems provide adaptive immunity in bacteria and archaea. Although there are two main classes of CRISPR–Cas systems defined by gene content, interfering RNA biogenesis, and effector proteins, Type II systems have recently been exploited on a broad scale to develop next-generation genetic engineering and genome-editing tools. Conveniently, Type II systems are streamlined and rely on a single protein, Cas9, and a guide RNA molecule, comprised of a CRISPR RNA (crRNA) and trans-acting CRISPR RNA (tracrRNA), to achieve effective and programmable nucleic acid targeting and cleavage. Currently, most commercially available Cas9-based genome-editing tools use the CRISPR–Cas system from Streptococcus pyogenes (SpyCas9), although many orthogonal Type II systems are available for diverse and multiplexable genome engineering applications. Here, we discuss the biological significance of Type II CRISPR–Cas elements, including the tracrRNA, crRNA, Cas9, and protospacer-adjacent motif (PAM), and look at the native function of these elements to understand how they can be engineered, enhanced, and optimized for genome editing applications. Additionally, we discuss the basis for orthogonal Cas9 and guide RNA systems that would allow researchers to concurrently use multiple Cas9-based systems for different purposes. Understanding the native function of endogenous Type II CRISPR–Cas systems can lead to new Cas9 tool development to expand the genetic manipulation toolbox.}, number={7}, journal={Cold Spring Harbor Protocols}, publisher={Cold Spring Harbor Laboratory}, author={Briner, Alexandra E. and Barrangou, Rodolphe}, year={2016}, month={Jul}, pages={pdb.top090902} }
 @article{leenay_maksimchuk_slotkowski_agrawal_gomaa_briner_barrangou_beisel_2016, title={Identifying and Visualizing Functional PAM Diversity across CRISPR-Cas Systems}, volume={62}, ISSN={["1097-4164"]}, DOI={10.1016/j.molcel.2016.02.031}, abstractNote={CRISPR-Cas adaptive immune systems in prokaryotes boast a diversity of protein families and mechanisms of action, where most systems rely on protospacer-adjacent motifs (PAMs) for DNA target recognition. Here, we developed an in vivo, positive, and tunable screen termed PAM-SCANR (PAM screen achieved by NOT-gate repression) to elucidate functional PAMs as well as an interactive visualization scheme termed the PAM wheel to convey individual PAM sequences and their activities. PAM-SCANR and the PAM wheel identified known functional PAMs while revealing complex sequence-activity landscapes for the Bacillus halodurans I-C (Cascade), Escherichia coli I-E (Cascade), Streptococcus thermophilus II-A CRISPR1 (Cas9), and Francisella novicida V-A (Cpf1) systems. The PAM wheel was also readily applicable to existing high-throughput screens and garnered insights into SpyCas9 and SauCas9 PAM diversity. These tools offer powerful means of elucidating and visualizing functional PAMs toward accelerating our ability to understand and exploit the multitude of CRISPR-Cas systems in nature.}, number={1}, journal={MOLECULAR CELL}, publisher={Elsevier BV}, author={Leenay, Ryan T. and Maksimchuk, Kenneth R. and Slotkowski, Rebecca A. and Agrawal, Roma N. and Gomaa, Ahmed A. and Briner, Alexandra E. and Barrangou, Rodolphe and Beisel, Chase L.}, year={2016}, month={Apr}, pages={137–147} }
 @article{brandt_barrangou_2016, title={Phylogenetic Analysis of the Bifidobacterium Genus Using Glycolysis Enzyme Sequences}, volume={7}, ISSN={["1664-302X"]}, DOI={10.3389/fmicb.2016.00657}, abstractNote={Bifidobacteria are important members of the human gastrointestinal tract that promote the establishment of a healthy microbial consortium in the gut of infants. Recent studies have established that the Bifidobacterium genus is a polymorphic phylogenetic clade, which encompasses a diversity of species and subspecies that encode a broad range of proteins implicated in complex and non-digestible carbohydrate uptake and catabolism, ranging from human breast milk oligosaccharides, to plant fibers. Recent genomic studies have created a need to properly place Bifidobacterium species in a phylogenetic tree. Current approaches, based on core-genome analyses come at the cost of intensive sequencing and demanding analytical processes. Here, we propose a typing method based on sequences of glycolysis genes and the proteins they encode, to provide insights into diversity, typing, and phylogeny in this complex and broad genus. We show that glycolysis genes occur broadly in these genomes, to encode the machinery necessary for the biochemical spine of the cell, and provide a robust phylogenetic marker. Furthermore, glycolytic sequences-based trees are congruent with both the classical 16S rRNA phylogeny, and core genome-based strain clustering. Furthermore, these glycolysis markers can also be used to provide insights into the adaptive evolution of this genus, especially with regards to trends toward a high GC content. This streamlined method may open new avenues for phylogenetic studies on a broad scale, given the widespread occurrence of the glycolysis pathway in bacteria, and the diversity of the sequences they encode.}, journal={FRONTIERS IN MICROBIOLOGY}, publisher={Frontiers Media SA}, author={Brandt, Katelyn and Barrangou, Rodolphe}, year={2016}, month={May} }
 @article{briner_henriksen_barrangou_2016, title={Prediction and Validation of Native and Engineered Cas9 Guide Sequences}, volume={2016}, DOI={10.1101/pdb.prot086785}, abstractNote={Cas9-based technologies rely on native elements of Type II CRISPR–Cas bacterial immune systems, including the trans-activating CRISPR RNA (tracrRNA), CRISPR RNA (crRNA), Cas9 protein, and protospacer-adjacent motif (PAM). The tracrRNA and crRNA form an RNA duplex that guides the Cas9 endonuclease to complementary nucleic acid sequences. Mechanistically, Cas9 initiates interactions by binding to the target PAM sequence and interrogating the target DNA in a 3′-to-5′ manner. Complementarity between the guide RNA and the target DNA is key. In natural systems, precise cleavage occurs when the target DNA sequence contains a PAM flanking a sequence homologous to the crRNA spacer sequence. Currently, the majority of commercial Cas9-based genome-editing tools are derived from the Type II CRISPR–Cas system of Streptococcus pyogenes. However, a diverse set of Type II CRISPR–Cas systems exist in nature that are potentially valuable for genome engineering applications. Exploitation of these systems requires prediction and validation of both native and engineered dual and single guide RNAs to drive Cas9 functionality. Here, we discuss how to identify the elements of these immune systems to develop next-generation Cas9-based genome-editing tools. We first discuss how to predict tracrRNA sequences and suggest a method for designing single guide RNAs containing only critical structural modules. We then outline how to predict the PAM sequence, which is crucial for determining potential targets for Cas9. Finally, validation of the system elements through transcriptome analysis and interference assays is essential for developing next-generation Cas9-based genome-editing tools.}, number={7}, journal={Cold Spring Harbor Protocols}, publisher={Cold Spring Harbor Laboratory}, author={Briner, Alexandra E. and Henriksen, Emily D. and Barrangou, Rodolphe}, year={2016}, month={Jul}, pages={pdb.prot086785} }
 @article{barrangou_birmingham_wiemann_beijersbergen_hornung_smith_2015, title={Advances in CRISPR-Cas9 genome engineering: lessons learned from RNA interference}, volume={43}, ISSN={["1362-4962"]}, DOI={10.1093/nar/gkv226}, abstractNote={The discovery that the machinery of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 bacterial immune system can be re-purposed to easily create deletions, insertions and replacements in the mammalian genome has revolutionized the field of genome engineering and re-invigorated the field of gene therapy. Many parallels have been drawn between the newly discovered CRISPR-Cas9 system and the RNA interference (RNAi) pathway in terms of their utility for understanding and interrogating gene function in mammalian cells. Given this similarity, the CRISPR-Cas9 field stands to benefit immensely from lessons learned during the development of RNAi technology. We examine how the history of RNAi can inform today's challenges in CRISPR-Cas9 genome engineering such as efficiency, specificity, high-throughput screening and delivery for in vivo and therapeutic applications.}, number={7}, journal={NUCLEIC ACIDS RESEARCH}, publisher={Oxford University Press (OUP)}, author={Barrangou, Rodolphe and Birmingham, Amanda and Wiemann, Stefan and Beijersbergen, Roderick L. and Hornung, Veit and Smith, Anja van Brabant}, year={2015}, month={Apr}, pages={3407–3419} }
 @misc{makarova_wolf_alkhnbashi_costa_shah_saunders_barrangou_brouns_charpentier_haft_et al._2015, title={An updated evolutionary classification of CRISPR-Cas systems}, volume={13}, number={11}, journal={Nature Reviews. Microbiology}, author={Makarova, K. S. and Wolf, Y. I. and Alkhnbashi, O. S. and Costa, F. and Shah, S. A. and Saunders, S. J. and Barrangou, R. and Brouns, S. J. J. and Charpentier, E. and Haft, D. H. and et al.}, year={2015}, pages={722–736} }
 @article{makarova_wolf_alkhnbashi_costa_shah_saunders_barrangou_brouns_charpentier_haft_et al._2015, title={An updated evolutionary classification of CRISPR–Cas systems}, volume={13}, ISSN={1740-1526 1740-1534}, url={http://dx.doi.org/10.1038/NRMICRO3569}, DOI={10.1038/nrmicro3569}, abstractNote={CRISPR–Cas systems provide bacteria and archaea with adaptive immunity to invading foreign DNA. In an Analysis article, Koonin and colleagues update a previous classification of these systems to incorporate the large volume of genomic data generated in recent years. The evolution of CRISPR–cas loci, which encode adaptive immune systems in archaea and bacteria, involves rapid changes, in particular numerous rearrangements of the locus architecture and horizontal transfer of complete loci or individual modules. These dynamics complicate straightforward phylogenetic classification, but here we present an approach combining the analysis of signature protein families and features of the architecture of cas loci that unambiguously partitions most CRISPR–cas loci into distinct classes, types and subtypes. The new classification retains the overall structure of the previous version but is expanded to now encompass two classes, five types and 16 subtypes. The relative stability of the classification suggests that the most prevalent variants of CRISPR–Cas systems are already known. However, the existence of rare, currently unclassifiable variants implies that additional types and subtypes remain to be characterized.}, number={11}, journal={Nature Reviews Microbiology}, publisher={Springer Science and Business Media LLC}, author={Makarova, Kira S. and Wolf, Yuri I. and Alkhnbashi, Omer S. and Costa, Fabrizio and Shah, Shiraz A. and Saunders, Sita J. and Barrangou, Rodolphe and Brouns, Stan J. J. and Charpentier, Emmanuelle and Haft, Daniel H. and et al.}, year={2015}, month={Sep}, pages={722–736} }
 @article{paez-espino_sharon_morovic_stahl_thomas_barrangou_banfield_2015, title={CRISPR Immunity Drives Rapid Phage Genome Evolution in Streptococcus thermophilus}, volume={6}, ISSN={["2150-7511"]}, DOI={10.1128/mbio.00262-15}, abstractNote={ABSTRACT
          
            Many bacteria rely on CRISPR-Cas systems to provide adaptive immunity against phages, predation by which can shape the ecology and functioning of microbial communities. To characterize the impact of CRISPR immunization on phage genome evolution, we performed long-term bacterium-phage (
            Streptococcus thermophilus
            -phage 2972) coevolution experiments. We found that in this species, CRISPR immunity drives fixation of single nucleotide polymorphisms that accumulate exclusively in phage genome regions targeted by CRISPR. Mutation rates in phage genomes highly exceed those of the host. The presence of multiple phages increased phage persistence by enabling recombination-based formation of chimeric phage genomes in which sequences heavily targeted by CRISPR were replaced. Collectively, our results establish CRISPR-Cas adaptive immunity as a key driver of phage genome evolution under the conditions studied and highlight the importance of multiple coexisting phages for persistence in natural systems.
          
          
            IMPORTANCE
            Phages remain an enigmatic part of the biosphere. As predators, they challenge the survival of host bacteria and archaea and set off an “arms race” involving host immunization countered by phage mutation. The CRISPR-Cas system is adaptive: by capturing fragments of a phage genome upon exposure, the host is positioned to counteract future infections. To investigate this process, we initiated massive deep-sequencing experiments with a host and infective phage and tracked the coevolution of both populations over hundreds of days. In the present study, we found that CRISPR immunity drives the accumulation of phage genome rearrangements (which enable longer phage survival) and escape mutations, establishing CRISPR as one of the fundamental drivers of phage evolution.
          }, number={2}, journal={MBIO}, publisher={American Society for Microbiology}, author={Paez-Espino, David and Sharon, Itai and Morovic, Wesley and Stahl, Buffy and Thomas, Brian C. and Barrangou, Rodolphe and Banfield, Jillian F.}, year={2015} }
 @article{selle_barrangou_2015, title={CRISPR-Based Technologies and the Future of Food Science}, volume={80}, DOI={10.1111/1750-3841.13094}, abstractNote={AbstractThe on‐going CRISPR craze is focused on the use of Cas9‐based technologies for genome editing applications in eukaryotes, with high potential for translational medicine and next‐generation gene therapy. Nevertheless, CRISPR‐Cas systems actually provide adaptive immunity in bacteria, and have much promise for various applications in food bacteria that include high‐resolution typing of pathogens, vaccination of starter cultures against phages, and the genesis of programmable and specific antibiotics that can selectively modulate bacterial population composition. Indeed, the molecular machinery from these DNA‐encoded, RNA‐mediated, DNA‐targeting systems can be harnessed in native hosts, or repurposed in engineered systems for a plethora of applications that can be implemented in all organisms relevant to the food chain, including agricultural crops trait‐enhancement, livestock breeding, and fermentation‐based manufacturing, and for the genesis of next‐generation food products with enhanced quality and health‐promoting functionalities. CRISPR‐based applications are now poised to revolutionize many fields within food science, from farm to fork. In this review, we describe CRISPR‐Cas systems and highlight their potential for the development of enhanced foods.}, number={11}, journal={Journal of Food Science}, publisher={Wiley-Blackwell}, author={Selle, Kurt and Barrangou, Rodolphe}, year={2015}, month={Oct}, pages={R2367–R2372} }
 @article{selle_klaenhammer_barrangou_2015, title={CRISPR-based screening of genomic island excision events in bacteria}, volume={112}, ISSN={["0027-8424"]}, DOI={10.1073/pnas.1508525112}, abstractNote={Significance
          The development of Clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated genes (CAS)–based technology for targeted genome editing has revolutionized molecular biology approaches, but significant and outstanding gaps exist for applications in bacteria, the native hosts of these adaptive immune systems. This study shows that CRISPR-Cas systems can be directed to target and delete genomic islands that are flanked by insertion-sequence elements and devoid of essential genes. Naturally occurring minor subpopulations harboring deletions in genomic islands were identified and readily isolated using CRISPR-Cas screening. Promising applications of this approach can define minimal bacterial genomes, determine essential genes, and characterize genetically heterogeneous bacterial populations.}, number={26}, journal={PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA}, publisher={Proceedings of the National Academy of Sciences}, author={Selle, Kurt and Klaenhammer, Todd R. and Barrangou, Rodolphe}, year={2015}, month={Jun}, pages={8076–8081} }
 @article{johnson_hymes_sanozky-dawes_henriksen_barrangou_klaenhammer_2016, title={Conserved S-Layer-Associated Proteins Revealed by Exoproteomic Survey of S-Layer-Forming Lactobacilli}, volume={82}, ISSN={["1098-5336"]}, url={https://doi.org/10.1128/AEM.01968-15}, DOI={10.1128/aem.01968-15}, abstractNote={ABSTRACT
          
            The
            Lactobacillus acidophilus
            homology group comprises Gram-positive species that include
            L. acidophilus
            ,
            L. helveticus
            ,
            L. crispatus
            ,
            L. amylovorus
            ,
            L. gallinarum
            ,
            L. delbrueckii
            subsp.
            bulgaricus
            ,
            L. gasseri
            , and
            L. johnsonii
            . While these bacteria are closely related, they have varied ecological lifestyles as dairy and food fermenters, allochthonous probiotics, or autochthonous commensals of the host gastrointestinal tract. Bacterial cell surface components play a critical role in the molecular dialogue between bacteria and interaction signaling with the intestinal mucosa. Notably, the
            L. acidophilus
            complex is distinguished in two clades by the presence or absence of S-layers, which are semiporous crystalline arrays of self-assembling proteinaceous subunits found as the outermost layer of the bacterial cell wall. In this study, S-layer-associated proteins (SLAPs) in the exoproteomes of various S-layer-forming
            Lactobacillus
            species were proteomically identified, genomically compared, and transcriptionally analyzed. Four gene regions encoding six putative SLAPs were conserved in the S-layer-forming
            Lactobacillus
            species but not identified in the extracts of the closely related progenitor,
            L. delbrueckii
            subsp.
            bulgaricus
            , which does not produce an S-layer. Therefore, the presence or absence of an S-layer has a clear impact on the exoproteomic composition of
            Lactobacillus
            species. This proteomic complexity and differences in the cell surface properties between S-layer- and non-S-layer-forming lactobacilli reveal the potential for SLAPs to mediate intimate probiotic interactions and signaling with the host intestinal mucosa.
          }, number={1}, journal={APPLIED AND ENVIRONMENTAL MICROBIOLOGY}, publisher={American Society for Microbiology}, author={Johnson, Brant R. and Hymes, Jeffrey and Sanozky-Dawes, Rosemary and Henriksen, Emily DeCrescenzo and Barrangou, Rodolphe and Klaenhammer, Todd R.}, editor={Nojiri, H.Editor}, year={2016}, month={Jan}, pages={134–145} }
 @article{barrangou_2015, title={Diversity of CRISPR-Cas immune systems and molecular machines}, volume={16}, DOI={10.1186/s13059-015-0816-9}, abstractNote={Bacterial adaptive immunity hinges on CRISPR-Cas systems that provide DNA-encoded, RNA-mediated targeting of exogenous nucleic acids. A plethora of CRISPR molecular machines occur broadly in prokaryotic genomes, with a diversity of Cas nucleases that can be repurposed for various applications.}, number={1}, journal={Genome Biology}, publisher={Springer Nature}, author={Barrangou, Rodolphe}, year={2015}, month={Nov} }
 @misc{barrangou_2015, title={Diversity of CRISPR-Cas immune systems and molecular machines}, volume={16}, journal={Genome Biology}, author={Barrangou, R.}, year={2015} }
 @article{sun_harris_mccann_guo_argimon_zhang_yang_jeffery_cooney_kagawa_et al._2015, title={Expanding the biotechnology potential of lactobacilli through comparative genomics of 213 strains and associated genera}, volume={6}, ISSN={["2041-1723"]}, DOI={10.1038/ncomms9322}, abstractNote={AbstractLactobacilli are a diverse group of species that occupy diverse nutrient-rich niches associated with humans, animals, plants and food. They are used widely in biotechnology and food preservation, and are being explored as therapeutics. Exploiting lactobacilli has been complicated by metabolic diversity, unclear species identity and uncertain relationships between them and other commercially important lactic acid bacteria. The capacity for biotransformations catalysed by lactobacilli is an untapped biotechnology resource. Here we report the genome sequences of 213 Lactobacillus strains and associated genera, and their encoded genetic catalogue for modifying carbohydrates and proteins. In addition, we describe broad and diverse presence of novel CRISPR-Cas immune systems in lactobacilli that may be exploited for genome editing. We rationalize the phylogenomic distribution of host interaction factors and bacteriocins that affect their natural and industrial environments, and mechanisms to withstand stress during technological processes. We present a robust phylogenomic framework of existing species and for classifying new species.}, journal={NATURE COMMUNICATIONS}, publisher={Springer Nature}, author={Sun, Zhihong and Harris, Hugh M. B. and McCann, Angela and Guo, Chenyi and Argimon, Silvia and Zhang, Wenyi and Yang, Xianwei and Jeffery, Ian B. and Cooney, Jakki C. and Kagawa, Todd F. and et al.}, year={2015}, month={Sep} }
 @misc{barrangou_pijkeren_2016, title={Exploiting CRISPR-Cas immune systems for genome editing in bacteria}, volume={37}, ISSN={["1879-0429"]}, DOI={10.1016/j.copbio.2015.10.003}, abstractNote={The CRISPR-Cas immune system is a DNA-encoded, RNA-mediated, DNA-targeting defense mechanism, which provides sequence-specific targeting of DNA. This molecular machinery can be engineered into the sgRNA:Cas9 technology, for programmable cleavage of DNA. Following the genesis of double-stranded DNA breaks, the DNA repair machinery generates mutations at the cleavage site using various pathways. This technology has revolutionized eukaryotic genome editing, and we are at the cusp of full exploitation in bacteria. Here, we discuss the potential of CRISPR-based technologies for use in bacteria, and highlight the application of single stranded DNA recombineering combined with CRISPR-Cas selection to edit the genome of a probiotic organism. We envision that CRISPR-Cas technologies will play a key role in the development of next-generation industrial bacteria.}, journal={CURRENT OPINION IN BIOTECHNOLOGY}, publisher={Elsevier BV}, author={Barrangou, Rodolphe and Pijkeren, Jan-Peter}, year={2016}, month={Feb}, pages={61–68} }
 @misc{selle_barrangou_2015, title={Harnessing CRISPR-Cas systems for bacterial genome editing}, volume={23}, ISSN={["1878-4380"]}, DOI={10.1016/j.tim.2015.01.008}, abstractNote={Manipulation of genomic sequences facilitates the identification and characterization of key genetic determinants in the investigation of biological processes. Genome editing via clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) constitutes a next-generation method for programmable and high-throughput functional genomics. CRISPR-Cas systems are readily reprogrammed to induce sequence-specific DNA breaks at target loci, resulting in fixed mutations via host-dependent DNA repair mechanisms. Although bacterial genome editing is a relatively unexplored and underrepresented application of CRISPR-Cas systems, recent studies provide valuable insights for the widespread future implementation of this technology. This review summarizes recent progress in bacterial genome editing and identifies fundamental genetic and phenotypic outcomes of CRISPR targeting in bacteria, in the context of tool development, genome homeostasis, and DNA repair.}, number={4}, journal={TRENDS IN MICROBIOLOGY}, publisher={Elsevier BV}, author={Selle, Kurt and Barrangou, Rodolphe}, year={2015}, month={Apr}, pages={225–232} }
 @article{selle_barrangou_2015, title={JFS Special Issue: 75 years of advancing food science, and preparing for the next 75 CRISPR-based technologies and the future of food science}, volume={80}, number={11}, journal={Journal of Food Science}, author={Selle, K. and Barrangou, R.}, year={2015}, pages={R2367–2372} }
 @article{sun_thomas_barrangou_banfield_2016, title={Metagenomic reconstructions of bacterial CRISPR loci constrain population histories}, volume={10}, ISSN={["1751-7370"]}, DOI={10.1038/ismej.2015.162}, abstractNote={Abstract
               Bacterial CRISPR-Cas systems provide insight into recent population history because they rapidly incorporate, in a unidirectional manner, short fragments (spacers) from coexisting infective virus populations into host chromosomes. Immunity is achieved by sequence identity between transcripts of spacers and their targets. Here, we used metagenomics to study the stability and dynamics of the type I-E CRISPR-Cas locus of Leptospirillum group II bacteria in biofilms sampled over 5 years from an acid mine drainage (AMD) system. Despite recovery of 452 686 spacers from CRISPR amplicons and metagenomic data, rarefaction curves of spacers show no saturation. The vast repertoire of spacers is attributed to phage/plasmid population diversity and retention of old spacers, despite rapid evolution of the targeted phage/plasmid genome regions (proto-spacers). The oldest spacers (spacers found at the trailer end) are conserved for at least 5 years, and 12% of these retain perfect or near-perfect matches to proto-spacer targets. The majority of proto-spacer regions contain an AAG proto-spacer adjacent motif (PAM). Spacers throughout the locus target the same phage population (AMDV1), but there are blocks of consecutive spacers without AMDV1 target sequences. Results suggest long-term coexistence of Leptospirillum with AMDV1 and periods when AMDV1 was less dominant. Metagenomics can be applied to millions of cells in a single sample to provide an extremely large spacer inventory, allow identification of phage/plasmids and enable analysis of previous phage/plasmid exposure. Thus, this approach can provide insights into prior bacterial environment and genetic interplay between hosts and their viruses.}, number={4}, journal={ISME JOURNAL}, publisher={Springer Nature}, author={Sun, Christine L. and Thomas, Brian C. and Barrangou, Rodolphe and Banfield, Jillian F.}, year={2016}, month={Apr}, pages={858–870} }
 @article{kyung_medina pradas_kim_lee_kim_choi_cho_chung_barrangou_breidt_2015, title={Microbial Ecology of Watery Kimchi}, volume={80}, ISSN={["1750-3841"]}, DOI={10.1111/1750-3841.12848}, abstractNote={AbstractThe biochemistry and microbial ecology of 2 similar types of watery (mul) kimchi, containing sliced and unsliced radish and vegetables (nabak and dongchimi, respectively), were investigated. Samples from kimchi were fermented at 4, 10, and 20 °C were analyzed by plating on differential and selective media, high‐performance liquid chromatography, and high‐throughput DNA sequencing of 16S rDNA. Nabak kimchi showed similar trends as dongchimi, with increasing lactic and acetic acids and decreasing pH for each temperature, but differences in microbiota were apparent. Interestingly, bacteria from the Proteobacterium phylum, including Enterobacteriaceae, decreased more rapidly during fermentation at 4 °C in nabak cabbage fermentations compared with dongchimi. Although changes for Proteobacterium and Enterobacteriaceae populations were similar during fermentation at 10 and 20 °C, the homolactic stage of fermentation did not develop for the 4 and 10 °C samples of both nabak and dongchimi during the experiment. These data show the differences in biochemistry and microbial ecology that can result from preparation method and fermentation conditions of the kimchi, which may impact safety (Enterobacteriaceae populations may include pathogenic bacteria) and quality (homolactic fermentation can be undesirable, if too much acid is produced) of the product. In addition, the data also illustrate the need for improved methods for identifying and differentiating closely related lactic acid bacteria species using high‐throughput sequencing methods.}, number={5}, journal={JOURNAL OF FOOD SCIENCE}, publisher={Wiley-Blackwell}, author={Kyung, Kyu Hang and Medina Pradas, Eduardo and Kim, Song Gun and Lee, Yong Jae and Kim, Kyong Ho and Choi, Jin Joo and Cho, Joo Hyong and Chung, Chang Ho and Barrangou, Rodolphe and Breidt, Frederick}, year={2015}, month={May}, pages={M1031–M1038} }
 @article{briner_lugli_milani_duranti_turroni_gueimonde_margolles_sinderen_ventura_barrangou_2015, title={Occurrence and Diversity of CRISPR-Cas Systems in the Genus Bifidobacterium}, volume={10}, ISSN={["1932-6203"]}, DOI={10.1371/journal.pone.0133661}, abstractNote={CRISPR-Cas systems constitute adaptive immune systems for antiviral defense in bacteria. We investigated the occurrence and diversity of CRISPR-Cas systems in 48 Bifidobacterium genomes to gain insights into the diversity and co-evolution of CRISPR-Cas systems within the genus and investigate CRISPR spacer content. We identified the elements necessary for the successful targeting and inference of foreign DNA in select Type II CRISPR-Cas systems, including the tracrRNA and target PAM sequence. Bifidobacterium species have a very high frequency of CRISPR-Cas occurrence (77%, 37 of 48). We found that many Bifidobacterium species have unusually large and diverse CRISPR-Cas systems that contain spacer sequences showing homology to foreign genetic elements like prophages. A large number of CRISPR spacers in bifidobacteria show perfect homology to prophage sequences harbored in the chromosomes of other species of Bifidobacterium, including some spacers that self-target the chromosome. A correlation was observed between strains that lacked CRISPR-Cas systems and the number of times prophages in that chromosome were targeted by other CRISPR spacers. The presence of prophage-targeting CRISPR spacers and prophage content may shed light on evolutionary processes and strain divergence. Finally, elements of Type II CRISPR-Cas systems, including the tracrRNA and crRNAs, set the stage for the development of genome editing and genetic engineering tools.}, number={7}, journal={PLOS ONE}, publisher={Public Library of Science (PLoS)}, author={Briner, Alexandra E. and Lugli, Gabriele Andrea and Milani, Christian and Duranti, Sabrina and Turroni, Francesca and Gueimonde, Miguel and Margolles, Abelardo and Sinderen, Douwe and Ventura, Marco and Barrangou, Rodolphe}, editor={Riedel, Christian U.Editor}, year={2015}, month={Jul} }
 @article{sanozky-dawes_selle_o'flaherty_klaenhammer_barrangou_2015, title={Occurrence and activity of a type II CRISPR-Cas system in Lactobacillus gasseri}, volume={161}, ISSN={["1350-0872"]}, DOI={10.1099/mic.0.000129}, abstractNote={Bacteria encode clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated genes (cas), which collectively form an RNA-guided adaptive immune system against invasive genetic elements. In silico surveys have revealed that lactic acid bacteria harbour a prolific and diverse set of CRISPR-Cas systems. Thus, the natural evolutionary role of CRISPR-Cas systems may be investigated in these ecologically, industrially, scientifically and medically important microbes. In this study, 17 Lactobacillus gasseri strains were investigated and 6 harboured a type II-A CRISPR-Cas system, with considerable diversity in array size and spacer content. Several of the spacers showed similarity to phage and plasmid sequences, which are typical targets of CRISPR-Cas immune systems. Aligning the protospacers facilitated inference of the protospacer adjacent motif sequence, determined to be 5'-NTAA-3' flanking the 3' end of the protospacer. The system in L. gasseri JV-V03 and NCK 1342 interfered with transforming plasmids containing sequences matching the most recently acquired CRISPR spacers in each strain. We report the distribution and function of a native type II-A CRISPR-Cas system in the commensal species L. gasseri. Collectively, these results open avenues for applications for bacteriophage protection and genome modification in L. gasseri, and contribute to the fundamental understanding of CRISPR-Cas systems in bacteria.}, journal={MICROBIOLOGY-SGM}, author={Sanozky-Dawes, Rosemary and Selle, Kurt and O'Flaherty, Sarah and Klaenhammer, Todd and Barrangou, Rodolphe}, year={2015}, month={Sep}, pages={1752–1761} }
 @misc{sontheimer_barrangou_2015, title={The Bacterial Origins of the CRISPR Genome-Editing Revolution}, volume={26}, ISSN={["1557-7422"]}, DOI={10.1089/hum.2015.091}, abstractNote={Like most of the tools that enable modern life science research, the recent genome-editing revolution has its biological roots in the world of bacteria and archaea. Clustered, regularly interspaced, short palindromic repeats (CRISPR) loci are found in the genomes of many bacteria and most archaea, and underlie an adaptive immune system that protects the host cell against invasive nucleic acids such as viral genomes. In recent years, engineered versions of these systems have enabled efficient DNA targeting in living cells from dozens of species (including humans and other eukaryotes), and the exploitation of the resulting endogenous DNA repair pathways has provided a route to fast, easy, and affordable genome editing. In only three years after RNA-guided DNA cleavage was first harnessed, the ability to edit genomes via simple, user-defined RNA sequences has already revolutionized nearly all areas of biological science. CRISPR-based technologies are now poised to similarly revolutionize many facets of clinical medicine, and even promise to advance the long-term goal of directly editing genomic sequences of patients with inherited disease. In this review, we describe the biological and mechanistic basis for these remarkable immune systems, and how their engineered derivatives are revolutionizing basic and clinical research.}, number={7}, journal={HUMAN GENE THERAPY}, publisher={Mary Ann Liebert Inc}, author={Sontheimer, Erik J. and Barrangou, Rodolphe}, year={2015}, month={Jul}, pages={413–424} }
 @misc{barrangou_2015, title={The roles of CRISPR-Cas systems in adaptive immunity and beyond}, volume={32}, ISSN={["1879-0372"]}, DOI={10.1016/j.coi.2014.12.008}, abstractNote={Clustered regularly interspaced short palindromic repeats (CRISPR) and accompanying Cas proteins constitute the adaptive CRISPR-Cas immune system in bacteria and archaea. This DNA-encoded, RNA-mediated defense system provides sequence-specific recognition, targeting and degradation of exogenous nucleic acid. Though the primary established role of CRISPR-Cas systems is in bona fide adaptive antiviral defense in bacteria, a growing body of evidence indicates that it also plays critical functional roles beyond immunity, such as endogenous transcriptional control. Furthermore, benefits inherent to maintaining genome homeostasis also come at the cost of reduced uptake of beneficial DNA, and preventing strategic adaptation to the environment. This opens new avenues for the investigation of CRISPR-Cas systems and their functional characterization beyond adaptive immunity.}, journal={CURRENT OPINION IN IMMUNOLOGY}, publisher={Elsevier BV}, author={Barrangou, Rodolphe}, year={2015}, month={Feb}, pages={36–41} }
 @article{beisel_gomaa_barrangou_2014, title={A CRISPR design for next-generation antimicrobials}, volume={15}, ISSN={["1474-760X"]}, DOI={10.1186/s13059-014-0516-x}, abstractNote={Two recent publications have demonstrated how delivering CRISPR nucleases provides a promising solution to the growing problem of bacterial antibiotic resistance.}, number={11}, journal={GENOME BIOLOGY}, publisher={Springer Nature}, author={Beisel, Chase L. and Gomaa, Ahmed A. and Barrangou, Rodolphe}, year={2014} }
 @article{barrangou_oost_2015, title={Bacteriophage exclusion, a new defense system}, volume={34}, ISSN={["1460-2075"]}, DOI={10.15252/embj.201490620}, abstractNote={The ability to withstand viral predation is critical for survival of most microbes. Accordingly, a plethora of phage resistance systems has been identified in bacterial genomes (Labrie et al, ), including restriction‐modification systems (R‐M) (Tock & Dryden, ), abortive infection (Abi) (Chopin et al, ), Argonaute‐based interference (Swarts et al, ), as well as clustered regularly interspaced short palindromic repeats (CRISPR) and associated protein (Cas) adaptive immune system (CRISPR‐Cas) (Barrangou & Marraffini, ; Van der Oost et al, ). Predictably, the dark matter of bacterial genomes contains a wealth of genetic gold. A study published in this issue of The EMBO Journal by Goldfarb et al ( ) unveils bacteriophage exclusion (BREX) as a novel, widespread bacteriophage resistance system that provides innate immunity against virulent and temperate phage in bacteria.}, number={2}, journal={EMBO JOURNAL}, publisher={EMBO}, author={Barrangou, Rodolphe and Oost, John}, year={2015}, month={Jan}, pages={134–135} }
 @misc{barrangou_marraffini_2014, title={CRISPR-Cas Systems: Prokaryotes Upgrade to Adaptive Immunity}, volume={54}, ISSN={["1097-4164"]}, DOI={10.1016/j.molcel.2014.03.011}, abstractNote={Clustered regularly interspaced short palindromic repeats (CRISPR), and associated proteins (Cas) comprise the CRISPR-Cas system, which confers adaptive immunity against exogenic elements in many bacteria and most archaea. CRISPR-mediated immunization occurs through the uptake of DNA from invasive genetic elements such as plasmids and viruses, followed by its integration into CRISPR loci. These loci are subsequently transcribed and processed into small interfering RNAs that guide nucleases for specific cleavage of complementary sequences. Conceptually, CRISPR-Cas shares functional features with the mammalian adaptive immune system, while also exhibiting characteristics of Lamarckian evolution. Because immune markers spliced from exogenous agents are integrated iteratively in CRISPR loci, they constitute a genetic record of vaccination events and reflect environmental conditions and changes over time. Cas endonucleases, which can be reprogrammed by small guide RNAs have shown unprecedented potential and flexibility for genome editing and can be repurposed for numerous DNA targeting applications including transcriptional control.}, number={2}, journal={MOLECULAR CELL}, publisher={Elsevier BV}, author={Barrangou, Rodolphe and Marraffini, Luciano A.}, year={2014}, month={Apr}, pages={234–244} }
 @article{barrangou_2014, title={Cas9 Targeting and the CRISPR Revolution}, volume={344}, ISSN={["1095-9203"]}, DOI={10.1126/science.1252964}, abstractNote={Uncovering how an RNA-protein molecular scalpel targets DNA will advance our ability to engineer genomes.}, number={6185}, journal={SCIENCE}, publisher={American Association for the Advancement of Science (AAAS)}, author={Barrangou, Rodolphe}, year={2014}, month={May}, pages={707–708} }
 @article{shariat_timme_pettengill_barrangou_dudley_2015, title={Characterization and evolution of Salmonella CRISPR-Cas systems}, volume={161}, ISSN={["1465-2080"]}, DOI={10.1099/mic.0.000005}, abstractNote={Prokaryotic CRISPR-Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated genes) systems provide adaptive immunity from invasive genetic elements and encompass three essential features: (i) cas genes, (ii) a CRISPR array composed of spacers and direct repeats and (iii) an AT-rich leader sequence upstream of the array. We performed in-depth sequence analysis of the CRISPR-Cas systems in >600 Salmonella, representing four clinically prevalent serovars. Each CRISPR-Cas feature is extremely conserved in the Salmonella, and the CRISPR1 locus is more highly conserved than CRISPR2. Array composition is serovar-specific, although no convincing evidence of recent spacer acquisition against exogenous nucleic acids exists. Only 12 % of spacers match phage and plasmid sequences and self-targeting spacers are associated with direct repeat variants. High nucleotide identity (>99.9 %) exists across the cas operon among isolates of a single serovar and in some cases this conservation extends across divergent serovars. These observations reflect historical CRISPR-Cas immune activity, showing that this locus has ceased undergoing adaptive events. Intriguingly, the high level of conservation across divergent serovars shows that the genetic integrity of these inactive loci is maintained over time, contrasting with the canonical view that inactive CRISPR loci degenerate over time. This thorough characterization of Salmonella CRISPR-Cas systems presents new insights into Salmonella CRISPR evolution, particularly with respect to cas gene conservation, leader sequences, organization of direct repeats and protospacer matches. Collectively, our data suggest that Salmonella CRISPR-Cas systems are no longer immunogenic; rather, their impressive conservation indicates they may have an alternative function in Salmonella.}, journal={MICROBIOLOGY-SGM}, author={Shariat, Nikki and Timme, Ruth E. and Pettengill, James B. and Barrangou, Rodolphe and Dudley, Edward G.}, year={2015}, month={Feb}, pages={374–386} }
 @article{barrangou_horvath_2014, title={Functions and Applications of RNA-Guided CRISPR-Cas Immune Systems}, DOI={10.1002/3527600906.mcb.20130001}, abstractNote={Clustered regularly interspaced short palindromic repeats (CRISPRs), together with CRISPR-associated sequences (cas) constitute the CRISPR-Cas adaptive immune system in bacteria and archaea. Adaptive immunity is built into CRISPR arrays through the uptake of small pieces of invasive nucleic acids, such as viruses and plasmids. Acquired immunity is subsequently mediated by small interfering RNAs transcribed from these loci, that guide specific cleavage of complementary sequences by nucleases. Studies have established that CRISPR loci and their RNA-guided interference machinery can be exploited for a broad array of applications. Adaptive immunity can be built against viruses, or to preclude the uptake of undesirable sequences. The inheritable and hypervariable nature of these loci can be used to track the phylogenetic path of an organism and reveal the evolutionary interplay between hosts and their viruses. Recently, a new customizable genome editing system was developed based on these versatile interfering RNAs to specifically guide nucleases for sequence cleavage. 
 
 
Keywords: 
 
Cas; 
Cas9; 
Cascade; 
CRISPR; 
crRNA; 
Interference}, journal={Encyclopedia of Molecular Cell Biology and Molecular Medicine}, author={Barrangou, Rodolphe and Horvath, Philippe}, year={2014}, month={Oct} }
 @article{milani_lugli_duranti_turroni_bottacini_mangifesta_sanchez_viappiani_mancabelli_taminiau_et al._2014, title={Genomic Encyclopedia of Type Strains of the Genus Bifidobacterium}, volume={80}, ISSN={["1098-5336"]}, DOI={10.1128/aem.02308-14}, abstractNote={ABSTRACT
          
            Bifidobacteria represent one of the dominant microbial groups that are present in the gut of various animals, being particularly prevalent during the suckling stage of life of humans and other mammals. However, the overall genome structure of this group of microorganisms remains largely unexplored. Here, we sequenced the genomes of 42 representative (sub)species across the
            Bifidobacterium
            genus and used this information to explore the overall genetic picture of this bacterial group. Furthermore, the genomic data described here were used to reconstruct the evolutionary development of the
            Bifidobacterium
            genus. This reconstruction suggests that its evolution was substantially influenced by genetic adaptations to obtain access to glycans, thereby representing a common and potent evolutionary force in shaping bifidobacterial genomes.
          }, number={20}, journal={APPLIED AND ENVIRONMENTAL MICROBIOLOGY}, publisher={American Society for Microbiology}, author={Milani, Christian and Lugli, Gabriele Andrea and Duranti, Sabrina and Turroni, Francesca and Bottacini, Francesca and Mangifesta, Marta and Sanchez, Borja and Viappiani, Alice and Mancabelli, Leonardo and Taminiau, Bernard and et al.}, year={2014}, month={Oct}, pages={6290–6302} }
 @article{briner_donohoue_gomaa_selle_slorach_nye_haurwitz_beisel_may_barrangou_2014, title={Guide RNA Functional Modules Direct Cas9 Activity and Orthogonality}, volume={56}, ISSN={["1097-4164"]}, DOI={10.1016/j.molcel.2014.09.019}, abstractNote={Highlights•Several modules within guide RNAs drive Cas9-mediated cleavage•These modules are universally relevant for Type II-A CRISPR-Cas systems•Guide RNAs can be altered to cross Cas9 orthogonality boundariesSummaryThe RNA-guided Cas9 endonuclease specifically targets and cleaves DNA in a sequence-dependent manner and has been widely used for programmable genome editing. Cas9 activity is dependent on interactions with guide RNAs, and evolutionarily divergent Cas9 nucleases have been shown to work orthogonally. However, the molecular basis of selective Cas9:guide-RNA interactions is poorly understood. Here, we identify and characterize six conserved modules within native crRNA:tracrRNA duplexes and single guide RNAs (sgRNAs) that direct Cas9 endonuclease activity. We show the bulge and nexus are necessary for DNA cleavage and demonstrate that the nexus and hairpins are instrumental in defining orthogonality between systems. In contrast, the crRNA:tracrRNA complementary region can be modified or partially removed. Collectively, our results establish guide RNA features that drive DNA targeting by Cas9 and open new design and engineering avenues for CRISPR technologies.Graphical abstract}, number={2}, journal={MOLECULAR CELL}, publisher={Elsevier BV}, author={Briner, Alexandra E. and Donohoue, Paul D. and Gomaa, Ahmed A. and Selle, Kurt and Slorach, Euan M. and Nye, Christopher H. and Haurwitz, Rachel E. and Beisel, Chase L. and May, Andrew P. and Barrangou, Rodolphe}, year={2014}, month={Oct}, pages={333–339} }
 @article{barrangou_klaenhammer_2014, title={Microbiology: Bacteria get vaccinated}, volume={513}, DOI={10.1038/513175a}, number={7517}, journal={Nature}, publisher={Springer Nature}, author={Barrangou, Rodolphe and Klaenhammer, Todd R.}, year={2014}, month={Sep}, pages={175–176} }
 @article{gomaa_klumpe_luo_selle_barrangou_beisel_2014, title={Programmable Removal of Bacterial Strains by Use of Genome-Targeting CRISPR-Cas Systems}, volume={5}, ISSN={["2150-7511"]}, DOI={10.1128/mbio.00928-13}, abstractNote={ABSTRACT
          
            CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems in bacteria and archaea employ CRISPR RNAs to specifically recognize the complementary DNA of foreign invaders, leading to sequence-specific cleavage or degradation of the target DNA. Recent work has shown that the accidental or intentional targeting of the bacterial genome is cytotoxic and can lead to cell death. Here, we have demonstrated that genome targeting with CRISPR-Cas systems can be employed for the sequence-specific and titratable removal of individual bacterial strains and species. Using the type I-E CRISPR-Cas system in
            Escherichia coli
            as a model, we found that this effect could be elicited using native or imported systems and was similarly potent regardless of the genomic location, strand, or transcriptional activity of the target sequence. Furthermore, the specificity of targeting with CRISPR RNAs could readily distinguish between even highly similar strains in pure or mixed cultures. Finally, varying the collection of delivered CRISPR RNAs could quantitatively control the relative number of individual strains within a mixed culture. Critically, the observed selectivity and programmability of bacterial removal would be virtually impossible with traditional antibiotics, bacteriophages, selectable markers, or tailored growth conditions. Once delivery challenges are addressed, we envision that this approach could offer a novel means to quantitatively control the composition of environmental and industrial microbial consortia and may open new avenues for the development of “smart” antibiotics that circumvent multidrug resistance and differentiate between pathogenic and beneficial microorganisms.
          
          
            IMPORTANCE
            Controlling the composition of microbial populations is a critical aspect in medicine, biotechnology, and environmental cycles. While different antimicrobial strategies, such as antibiotics, antimicrobial peptides, and lytic bacteriophages, offer partial solutions, what remains elusive is a generalized and programmable strategy that can distinguish between even closely related microorganisms and that allows for fine control over the composition of a microbial population. This study demonstrates that RNA-directed immune systems in bacteria and archaea called CRISPR-Cas systems can provide such a strategy. These systems can be employed to selectively and quantitatively remove individual bacterial strains based purely on sequence information, creating opportunities in the treatment of multidrug-resistant infections, the control of industrial fermentations, and the study of microbial consortia.
          }, number={1}, journal={MBIO}, publisher={American Society for Microbiology}, author={Gomaa, Ahmed A. and Klumpe, Heidi E. and Luo, Michelle L. and Selle, Kurt and Barrangou, Rodolphe and Beisel, Chase L.}, year={2014} }
 @article{horvath_barrangou, title={Protection against Foreign DNA}, DOI={10.1128/9781555816841.ch19}, abstractNote={This chapter briefly addresses the well-characterized restriction-modification system (R-M), non-sugar-specific nucleases (SNSN), and histone-like nucleoid structuring (H-NS). It more specifically elaborates on clustered regularly interspaced short palindromic repeats (CRISPR). CRISPR/CRISPR-associated (Cas), a recently described microbial system, provides acquired immunity against phages and plasmids by targeting nucleic acids in a sequence-specific manner. CRISPR features may be exploited for typing purposes, ecological and epidemiological studies, and also for enhancing phage resistance in bacteria. R-M systems commonly act as the first line of intracellular defense against foreign DNA. Some SNSN, such as Vvn from Vibrio vulnificus and EndoI from Escherichia coli, are periplasmic and thus prevent the uptake of foreign DNA. The ubiquitous and predatory nature of phages may explain the overwhelming representation of phage sequences in CRISPR spacers, but a recent report showed that CRISPR can dramatically impact the ability of plasmids to transfer genetic material in Staphylococcus epidermidis. Also, this study experimentally confirmed that CRISPR targets DNA directly in Staphylococcus. The CRISPR RNAs (crRNAs) seem to specifically guide the Cas defense apparatus toward foreign nucleic acid molecules that match the sequence of the spacers. This study also showed that Cas3, a predicted HD nuclease fused to a DEAD-box helicase, is required for the phage-resistance phenotype. The extent of the impact of CRISPR on phage genomes is perhaps best illustrated by extensive genome recombination events observed in environmental phage populations in response to CRISPR.}, journal={Bacterial Stress Responses, Second Edition}, publisher={American Society of Microbiology}, author={Horvath, Philippe and Barrangou, Rodolphe}, pages={333–348} }
 @article{wehnes_rehberger_barrangou_smith_2014, title={Short communication: Determination of Salmonella clustered regularly interspaced short palindromic repeats (CRISPR) diversity on dairy farms in Wisconsin and Minnesota}, volume={97}, DOI={10.3168/jds.2013-7595}, abstractNote={Salmonella enterica ssp. enterica is a foodborne pathogen able to cause disease in both humans and animals. Diverse serovars of this pathogen exist, some of which are host specific, causing a range of clinical symptoms from asymptomatic infection through morbidity and mortality. According to a 2007 survey by the USDA National Animal Health Monitoring System, fecal shedding of Salmonella from healthy cows occurs on 39.7% of dairy farms in the United States. Certain serovars are frequently isolated from dairy farms and the majority of isolates from the National Animal Health Monitoring System study were represented by 5 serovars; however, genotypic diversity was not examined. The objective of this study was to determine the diversity of clustered regularly interspaced short palindromic repeats (CRISPR) loci in Salmonella collected from 8 dairy farms with a previous history of salmonellosis. None of the cows or calves sampled on 2 of the 8 dairy farms were shedding Salmonella, although Salmonella was detected in a cow bedding sample on 1 of these farms. Salmonella populations were discrete on each farm, according to CRISPR typing, with the exception of an Anatum var. 15+ type on farms 5 and 6 and the Montevideo type on farms 1 and 2. One to 4 distinct CRISPR genotypes were identified per farm. The CRISPR typing differed within serovars, as Montevideo, Anatum var. 15+, and Muenster serovars had no overlap of spacer content, even on the same farm, reflecting between- and within-serovar genetic diversity. The dynamic nature of Salmonella populations was shown in a farm that was sampled longitudinally over 13.5 mo. Changes in serovar from 3,19:-:z27 to Montevideo was observed between the first sampling time and 8 mo later, with concomitant change in CRISPR alleles. The results indicate that Salmonella strains present in smaller dairy herds (<500 head) are specific to that farm and new Salmonella strains may emerge over time.}, number={10}, journal={Journal of Dairy Science}, publisher={American Dairy Science Association}, author={Wehnes, C.A. and Rehberger, T.G. and Barrangou, R. and Smith, A.H.}, year={2014}, month={Oct}, pages={6370–6377} }
 @article{pettengill_timme_barrangou_toro_allard_strain_musser_brown_2014, title={The evolutionary history and diagnostic utility of the CRISPR-Cas system within Salmonella enterica ssp enterica}, volume={2}, ISSN={["2167-8359"]}, DOI={10.7717/peerj.340}, abstractNote={Evolutionary studies of clustered regularly interspaced short palindromic repeats (CRISPRs) and their associated (cas) genes can provide insights into host-pathogen co-evolutionary dynamics and the frequency at which different genomic events (e.g., horizontal vs. vertical transmission) occur. Within this study, we used whole genome sequence (WGS) data to determine the evolutionary history and genetic diversity of CRISPR loci and cas genes among a diverse set of 427 Salmonella enterica ssp. enterica isolates representing 64 different serovars. We also evaluated the performance of CRISPR loci for typing when compared to whole genome and multilocus sequence typing (MLST) approaches. We found that there was high diversity in array length within both CRISPR1 (median = 22; min = 3; max = 79) and CRISPR2 (median = 27; min = 2; max = 221). There was also much diversity within serovars (e.g., arrays differed by as many as 50 repeat-spacer units among Salmonella ser. Senftenberg isolates). Interestingly, we found that there are two general cas gene profiles that do not track phylogenetic relationships, which suggests that non-vertical transmission events have occurred frequently throughout the evolutionary history of the sampled isolates. There is also considerable variation among the ranges of pairwise distances estimated within each cas gene, which may be indicative of the strength of natural selection acting on those genes. We developed a novel clustering approach based on CRISPR spacer content, but found that typing based on CRISPRs was less accurate than the MLST-based alternative; typing based on WGS data was the most accurate. Notwithstanding cost and accessibility, we anticipate that draft genome sequencing, due to its greater discriminatory power, will eventually become routine for traceback investigations.}, journal={PEERJ}, publisher={PeerJ}, author={Pettengill, James B. and Timme, Ruth E. and Barrangou, Rodolphe and Toro, Magaly and Allard, Marc W. and Strain, Errol and Musser, Steven M. and Brown, Eric W.}, year={2014}, month={Apr} }
 @article{carte_christopher_smith_olson_barrangou_moineau_glover_graveley_terns_terns_2014, title={The three major types of CRISPR-Cas systems function independently in CRISPR RNA biogenesis in Streptococcus thermophilus}, volume={93}, ISSN={["1365-2958"]}, DOI={10.1111/mmi.12644}, abstractNote={SummaryCRISPR‐Cas systems are small RNA‐based immune systems that protect prokaryotes from invaders such as viruses and plasmids. We have investigated the features and biogenesis of the CRISPR (cr)RNAs in Streptococcus thermophilus (Sth) strain DGCC7710, which possesses four different CRISPR‐Cas systems including representatives from the three major types of CRISPR‐Cas systems. Our results indicate that the crRNAs from each CRISPR locus are specifically processed into divergent crRNA species by Cas proteins (and non‐coding RNAs) associated with the respective locus. We find that the Csm Type III‐A and Cse Type I–E crRNAs are specifically processed by Cas6 and Cse3 (Cas6e), respectively, and retain an 8‐nucleotide CRISPR repeat sequence tag 5′ of the invader‐targeting sequence. The Cse Type I–E crRNAs also retain a 21‐nucleotide 3′ repeat tag. The crRNAs from the two Csn Type II‐A systems in Sth consist of a 5′‐truncated targeting sequence and a 3′ tag; however, these are distinct in size between the two. Moreover, the Csn1 (Cas9) protein associated with one Csn locus functions specifically in the production of crRNAs from that locus. Our findings indicate that multiple CRISPR‐Cas systems can function independently in crRNA biogenesis within a given organism – an important consideration in engineering coexisting CRISPR‐Cas pathways.}, number={1}, journal={MOLECULAR MICROBIOLOGY}, publisher={Wiley-Blackwell}, author={Carte, Jason and Christopher, Ross T. and Smith, Justin T. and Olson, Sara and Barrangou, Rodolphe and Moineau, Sylvain and Glover, Claiborne V. C., III and Graveley, Brenton R. and Terns, Rebecca M. and Terns, Michael P.}, year={2014}, month={Jul}, pages={98–112} }
 @article{barrangou_may_2015, title={Unraveling the potential of CRISPR-Cas9 for gene therapy}, volume={15}, ISSN={["1744-7682"]}, DOI={10.1517/14712598.2015.994501}, abstractNote={The molecular machinery from the prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)-Cas immune system has broadly been repurposed for genome editing in eukaryotes. In particular, the sequence-specific Cas9 endonuclease can be flexibly harnessed for the genesis of precise double-stranded DNA breaks, using single guide RNAs that are readily programmable. The endogenous DNA repair machinery subsequently generates genome modifications, either by random insertion or deletions using non-homologous end joining (NHEJ), or designed integration of mutations or genetic material using homology-directed repair (HDR) templates. This technology has opened new avenues for the investigation of genetic diseases in general, and for gene therapy applications in particular.}, number={3}, journal={EXPERT OPINION ON BIOLOGICAL THERAPY}, publisher={Informa Healthcare}, author={Barrangou, Rodolphe and May, Andrew P.}, year={2015}, month={Mar}, pages={311–314} }
 @article{hachem_møller_andersen_fredslund_majumder_nakai_leggio_goh_barrangou_klaenhammer_et al._2013, title={A Snapshot into the Metabolism of Isomalto-oligosaccharides in Probiotic Bacteria}, volume={60}, DOI={10.5458/jag.jag.jag-2012_022}, abstractNote={In vitro and in vivo studies have demonstrated the prebiotic potential of isomalto-oligosaccharides (IMO), comprising α -(1,6)-gluco-oligosaccharides and panose, which selectively stimulate the growth of probiotic bifidobacteria and lactobacilli. The protein machinery conferring the utilization of IMO by probiotics, however, remains vaguely described. We have used genomic, transcriptomic, enzymatic, and biophysical analyses to explore IMO utilization routes in probiotic lactobacilli and bifidobacteria as represented by Lactobacillus acidophilus NCFM and Bifidobacterium animalis subsp. lactis Bl-04, respectively. Utilization of IMO and malto-oligosaccharide ( α -(1,4)-glucosides) appears to be linked both at the genetic and transcriptomic level in the acidophilus group lactobacilli as suggested by reverse transcriptase PCR (RT-PCR) and gene landscape analysis. Canonical intracellular GH13_31 glucan 1,6- α -glucosidases active on IMO longer than isomaltose occur widely in acidophilus group lactobacilli. Interestingly, however, isomaltose, isomaltulose and panose seem to be internalized through a phosphoenoyl pyruvate transferase system (PTS) and subsequently hydrolyzed by a GH4 6-phosphate- α -glucosidases based on whole genome microarray analysis. This sub-specificity within GH4 seems to be unique for lactobacilli mainly from the gut niche, as the sequences from this group segregate from characterized GH4 maltose-6-phosphate- α -glucosidases in other organisms. By comparison, IMO is linked α -galactosidases, GH13_31 oligo 1,6- α -glucosidases and a dual specificity ATP-binding cassette (ABC) transport system active in the uptake of both classes of α -(1,6) glycosides. These data bring novel insight to advance our understanding of the basis of selectivity of IMO metabolism by important gut microbiome members.}, number={2}, journal={Journal of Applied Glycoscience}, publisher={The Japanese Society of Applied Glycoscience}, author={Hachem, Maher Abou and Møller, Marie S. and Andersen, Joakim M. and Fredslund, Folmer and Majumder, Avishek and Nakai, Hiroyuki and Leggio, Leila Lo and Goh, Yong-Jun and Barrangou, Rodolphe and Klaenhammer, Todd R. and et al.}, year={2013}, pages={95–100} }
 @article{dimarzio_shariat_kariyawasam_barrangou_dudley_2013, title={Antibiotic Resistance in Salmonella enterica Serovar Typhimurium Associates with CRISPR Sequence Type}, volume={57}, ISSN={["1098-6596"]}, DOI={10.1128/aac.00913-13}, abstractNote={ABSTRACT
          
            Salmonella enterica
            subsp.
            enterica
            serovar Typhimurium is a leading cause of food-borne salmonellosis in the United States. The number of antibiotic-resistant isolates identified in humans is steadily increasing, suggesting that the spread of antibiotic-resistant strains is a major threat to public health.
            S
            . Typhimurium is commonly identified in a wide range of animal hosts, food sources, and environments, but little is known about the factors mediating the spread of antibiotic resistance in this ecologically complex serovar. Previously, we developed a subtyping method, CRISPR–multi-virulence-locus sequence typing (MVLST), which discriminates among strains of several common
            S. enterica
            serovars. Here, CRISPR-MVLST identified 22 sequence types within a collection of 76
            S
            . Typhimurium isolates from a variety of animal sources throughout central Pennsylvania. Six of the sequence types were identified in more than one isolate, and we observed statistically significant differences in resistance among these sequence types to 7 antibiotics commonly used in veterinary and human medicine, such as ceftiofur and ampicillin (
            P
            < 0.05). Importantly, five of these sequence types were subsequently identified in human clinical isolates, and a subset of these isolates had identical antibiotic resistance patterns, suggesting that these subpopulations are being transmitted through the food system. Therefore, CRISPR-MVLST is a promising subtyping method for monitoring the farm-to-fork spread of antibiotic resistance in
            S
            . Typhimurium.
          }, number={9}, journal={ANTIMICROBIAL AGENTS AND CHEMOTHERAPY}, publisher={American Society for Microbiology}, author={DiMarzio, Michael and Shariat, Nikki and Kariyawasam, Subhashinie and Barrangou, Rodolphe and Dudley, Edward G.}, year={2013}, month={Sep}, pages={4282–4289} }
 @article{toro_cao_ju_allard_barrangou_zhao_brown_meng_2014, title={Association of Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) Elements with Specific Serotypes and Virulence Potential of Shiga Toxin-Producing Escherichia coli}, volume={80}, ISSN={["1098-5336"]}, DOI={10.1128/aem.03018-13}, abstractNote={ABSTRACT
          
            Shiga toxin-producing
            Escherichia coli
            (STEC) strains (
            n
            = 194) representing 43 serotypes and
            E. coli
            K-12 were examined for clustered regularly interspaced short palindromic repeat (CRISPR) arrays to study genetic relatedness among STEC serotypes. A subset of the strains (
            n
            = 81) was further analyzed for subtype I-E
            cas
            and virulence genes to determine a possible association of CRISPR elements with potential virulence. Four types of CRISPR arrays were identified. CRISPR1 and CRISPR2 were present in all strains tested; 1 strain also had both CRISPR3 and CRISPR4, whereas 193 strains displayed a short, combined array, CRISPR3-4. A total of 3,353 spacers were identified, representing 528 distinct spacers. The average length of a spacer was 32 bp. Approximately one-half of the spacers (54%) were unique and found mostly in strains of less common serotypes. Overall, CRISPR spacer contents correlated well with STEC serotypes, and identical arrays were shared between strains with the same H type (O26:H11, O103:H11, and O111:H11). There was no association identified between the presence of subtype I-E
            cas
            and virulence genes, but the total number of spacers had a negative correlation with potential pathogenicity (
            P
            < 0.05). Fewer spacers were found in strains that had a greater probability of causing outbreaks and disease than in those with lower virulence potential (
            P
            < 0.05). The relationship between the CRISPR-
            cas
            system and potential virulence needs to be determined on a broader scale, and the biological link will need to be established.
          }, number={4}, journal={APPLIED AND ENVIRONMENTAL MICROBIOLOGY}, publisher={American Society for Microbiology}, author={Toro, Magaly and Cao, Guojie and Ju, Wenting and Allard, Marc and Barrangou, Rodolphe and Zhao, Shaohua and Brown, Eric and Meng, Jianghong}, year={2014}, month={Feb}, pages={1411–1420} }
 @article{loquasto_barrangou_dudley_stahl_chen_roberts_2013, title={Bifidobacterium animalis subsp lactis ATCC 27673 Is a Genomically Unique Strain within Its Conserved Subspecies}, volume={79}, ISSN={["1098-5336"]}, DOI={10.1128/aem.01777-13}, abstractNote={ABSTRACT
          
            Many strains of
            Bifidobacterium animalis
            subsp.
            lactis
            are considered health-promoting probiotic microorganisms and are commonly formulated into fermented dairy foods. Analyses of previously sequenced genomes of
            B. animalis
            subsp.
            lactis
            have revealed little genetic diversity, suggesting that it is a monomorphic subspecies. However, during a multilocus sequence typing survey of
            Bifidobacterium
            , it was revealed that
            B. animalis
            subsp.
            lactis
            ATCC 27673 gave a profile distinct from that of the other strains of the subspecies. As part of an ongoing study designed to understand the genetic diversity of this subspecies, the genome of this strain was sequenced and compared to other sequenced genomes of
            B. animalis
            subsp.
            lactis
            and
            B. animalis
            subsp.
            animalis
            . The complete genome of ATCC 27673 was 1,963,012 bp, contained 1,616 genes and 4 rRNA operons, and had a G+C content of 61.55%. Comparative analyses revealed that the genome of ATCC 27673 contained six distinct genomic islands encoding 83 open reading frames not found in other strains of the same subspecies. In four islands, either phage or mobile genetic elements were identified. In island 6, a novel clustered regularly interspaced short palindromic repeat (CRISPR) locus which contained 81 unique spacers was identified. This type I-E CRISPR-
            cas
            system differs from the type I-C systems previously identified in this subspecies, representing the first identification of a different system in
            B. animalis
            subsp.
            lactis
            . This study revealed that ATCC 27673 is a strain of
            B. animalis
            subsp.
            lactis
            with novel genetic content and suggests that the lack of genetic variability observed is likely due to the repeated sequencing of a limited number of widely distributed commercial strains.
          }, number={22}, journal={APPLIED AND ENVIRONMENTAL MICROBIOLOGY}, publisher={American Society for Microbiology}, author={Loquasto, Joseph R. and Barrangou, Rodolphe and Dudley, Edward G. and Stahl, Buffy and Chen, Chun and Roberts, Robert F.}, year={2013}, month={Nov}, pages={6903–6910} }
 @book{barrangou_oost_2013, title={CRISPR-Cas Systems}, DOI={10.1007/978-3-662-45794-8}, publisher={Springer Berlin Heidelberg}, year={2013} }
 @misc{barrangou_2013, title={CRISPR-Cas systems and RNA-guided interference}, volume={4}, ISSN={["1757-7012"]}, DOI={10.1002/wrna.1159}, abstractNote={AbstractClustered regularly interspaced short palindromic repeats (CRISPR) together with associated sequences (cas) form the CRISPR‐Cas system, which provides adaptive immunity against viruses and plasmids in bacteria and archaea. Immunity is built through acquisition of short stretches of invasive nucleic acids into CRISPR loci as ‘spacers'. These immune markers are transcribed and processed into small noncoding interfering CRISPR RNAs (crRNAs) that guide Cas proteins toward target nucleic acids for specific cleavage of homologous sequences. Mechanistically, CRISPR‐Cas systems function in three distinct stages, namely: (1) adaptation, where new spacers are acquired from invasive elements for immunization; (2) crRNA biogenesis, where CRISPR loci are transcribed and processed into small interfering crRNAs; and (3) interference, where crRNAs guide the Cas machinery to specifically cleave homologous invasive nucleic acids. A number of studies have shown that CRISPR‐mediated immunity can readily increase the breadth and depth of virus resistance in bacteria and archaea. CRISPR interference can also target plasmid sequences and provide a barrier against the uptake of undesirable mobile genetic elements. These inheritable hypervariable loci provide phylogenetic information that can be insightful for typing purposes, epidemiological studies, and ecological surveys of natural habitats and environmental samples. More recently, the ability to reprogram CRISPR‐directed endonuclease activity using customizable small noncoding interfering RNAs has set the stage for novel genome editing and engineering avenues. This review highlights recent studies that revealed the molecular basis of CRISPR‐mediated immunity, and discusses applications of crRNA‐guided interference. WIREs RNA 2013, 4:267–278. doi: 10.1002/wrna.1159This article is categorized under:

RNA Turnover and Surveillance > Turnover/Surveillance Mechanisms
Regulatory RNAs/RNAi/Riboswitches > Biogenesis of Effector Small RNAs
Regulatory RNAs/RNAi/Riboswitches > RNAi: Mechanisms of Action
}, number={3}, journal={WILEY INTERDISCIPLINARY REVIEWS-RNA}, publisher={Wiley-Blackwell}, author={Barrangou, Rodolphe}, year={2013}, pages={267–278} }
 @article{shariat_sandt_dimarzio_barrangou_dudley_2013, title={CRISPR-MVLST subtyping of Salmonella enterica subsp. enterica serovars Typhimurium and Heidelberg and application in identifying outbreak isolates}, volume={13}, DOI={10.1186/1471-2180-13-254}, abstractNote={Abstract
          
            Background
            
              Salmonella enterica subsp. enterica serovars Typhimurium (S. Typhimurium) and Heidelberg (S. Heidelberg) are major causes of foodborne salmonellosis, accounting for a fifth of all annual salmonellosis cases in the United States. Rapid, efficient and accurate methods for identification are required for routine surveillance and to track specific strains during outbreaks. We used Pulsed-field Gel Electrophoresis (PFGE) and a recently developed molecular subtyping approach termed CRISPR-MVLST that exploits the hypervariable nature of virulence genes and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) to subtype clinical S. Typhimurium and S. Heidelberg isolates.
          
          
            Results
            We analyzed a broad set of 175 S. Heidelberg and S. Typhimurium isolates collected over a five-year period. We identified 21 Heidelberg Sequence Types (HSTs) and 37 Typhimurium STs (TSTs) that were represented by 27 and 45 PFGE pulsotypes, respectively, and determined the discriminatory power of each method.
          
          
            Conclusions
            For S. Heidelberg, our data shows that combined typing by both CRISPR-MVLST and PFGE provided a discriminatory power of 0.9213. Importantly, CRISPR-MVLST was able to separate common PFGE patterns such as JF6X01.0022 into distinct STs, thus providing significantly greater discriminatory power. Conversely, we show that subtyping by either CRISPR-MVLST or PFGE independently provides a sufficient discriminatory power (0.9345 and 0.9456, respectively) for S. Typhimurium. Additionally, using isolates from two S. Typhimurium outbreaks, we demonstrate that CRISPR-MVLST provides excellent epidemiologic concordance.
          }, number={1}, journal={BMC Microbiology}, publisher={Springer Nature}, author={Shariat, Nikki and Sandt, Carol H and DiMarzio, Michael J and Barrangou, Rodolphe and Dudley, Edward G}, year={2013}, pages={254} }
 @article{stahl_barrangou_2013, title={Complete Genome Sequence of Probiotic Strain Lactobacillus acidophilus La-14}, volume={1}, DOI={10.1128/genomea.00376-13}, abstractNote={ABSTRACT
          
            We present the 1,991,830-bp complete genome sequence of
            Lactobacillus acidophilus
            strain La-14 (SD-5212). Comparative genomic analysis revealed 99.98% similarity overall to the
            L. acidophilus
            NCFM genome. Globally, 111 single nucleotide polymorphisms (SNPs) (95 SNPs, 16 indels) were observed throughout the genome. Also, a 416-bp deletion in the LA14_1146 sugar ABC transporter was identified.
          }, number={3}, journal={Genome Announcements}, publisher={American Society for Microbiology}, author={Stahl, B. and Barrangou, R.}, year={2013}, month={Jun}, pages={e00376–13-e00376–13} }
 @article{barrangou_coute-monvoisin_stahl_chavichvily_damange_romero_boyaval_fremaux_horvath_2013, title={Genomic impact of CRISPR immunization against bacteriophages}, volume={41}, ISSN={["1470-8752"]}, DOI={10.1042/bst20130160}, abstractNote={CRISPR (clustered regularly interspaced short palindromic repeats) together with cas (CRISPR-associated) genes form the CRISPR–Cas immune system, which provides sequence-specific adaptive immunity against foreign genetic elements in bacteria and archaea. Immunity is acquired by the integration of short stretches of invasive DNA as novel ‘spacers’ into CRISPR loci. Subsequently, these immune markers are transcribed and generate small non-coding interfering RNAs that specifically guide nucleases for sequence-specific cleavage of complementary sequences. Among the four CRISPR–Cas systems present in Streptococcus thermophilus, CRISPR1 and CRISPR3 have the ability to readily acquire new spacers following bacteriophage or plasmid exposure. In order to investigate the impact of building CRISPR-encoded immunity on the host chromosome, we determined the genome sequence of a BIM (bacteriophage-insensitive mutant) derived from the DGCC7710 model organism, after four consecutive rounds of bacteriophage challenge. As expected, active CRISPR loci evolved via polarized addition of several novel spacers following exposure to bacteriophages. Although analysis of the draft genome sequence revealed a variety of SNPs (single nucleotide polymorphisms) and INDELs (insertions/deletions), most of the in silico differences were not validated by Sanger re-sequencing. In addition, two SNPs and two small INDELs were identified and tracked in the intermediate variants. Overall, building CRISPR-encoded immunity does not significantly affect the genome, which allows the maintenance of important functional properties in isogenic CRISPR mutants. This is critical for the development and formulation of sustainable and robust next-generation starter cultures with increased industrial lifespans.}, number={6}, journal={BIOCHEMICAL SOCIETY TRANSACTIONS}, publisher={Portland Press Ltd.}, author={Barrangou, Rodolphe and Coute-Monvoisin, Anne-Claire and Stahl, Buffy and Chavichvily, Isabelle and Damange, Florian and Romero, Dennis A. and Boyaval, Patrick and Fremaux, Christophe and Horvath, Philippe}, year={2013}, month={Dec}, pages={1383–1391} }
 @article{sinkunas_gasiunas_waghmare_dickman_barrangou_horvath_siksnys_2013, title={In vitro reconstitution of Cascade-mediated CRISPR immunity in Streptococcus thermophilus}, volume={32}, DOI={10.1038/emboj.2012.352}, abstractNote={Article18 January 2013free access Source Data In vitro reconstitution of Cascade-mediated CRISPR immunity in Streptococcus thermophilus Tomas Sinkunas Tomas Sinkunas Department of Protein–DNA Interactions, Institute of Biotechnology, Vilnius University, Vilnius, Lithuania Search for more papers by this author Giedrius Gasiunas Giedrius Gasiunas Department of Protein–DNA Interactions, Institute of Biotechnology, Vilnius University, Vilnius, Lithuania Search for more papers by this author Sakharam P Waghmare Sakharam P Waghmare Department of Chemical and Biological Engineering, ChELSI Institute, University of Sheffield, Sheffield, UK Search for more papers by this author Mark J Dickman Mark J Dickman Department of Chemical and Biological Engineering, ChELSI Institute, University of Sheffield, Sheffield, UK Search for more papers by this author Rodolphe Barrangou Rodolphe Barrangou DuPont Nutrition and Health, Madison, WI, USA Search for more papers by this author Philippe Horvath Philippe Horvath DuPont Nutrition and Health, Dangé-Saint-Romain, France Search for more papers by this author Virginijus Siksnys Corresponding Author Virginijus Siksnys Department of Protein–DNA Interactions, Institute of Biotechnology, Vilnius University, Vilnius, Lithuania Search for more papers by this author Tomas Sinkunas Tomas Sinkunas Department of Protein–DNA Interactions, Institute of Biotechnology, Vilnius University, Vilnius, Lithuania Search for more papers by this author Giedrius Gasiunas Giedrius Gasiunas Department of Protein–DNA Interactions, Institute of Biotechnology, Vilnius University, Vilnius, Lithuania Search for more papers by this author Sakharam P Waghmare Sakharam P Waghmare Department of Chemical and Biological Engineering, ChELSI Institute, University of Sheffield, Sheffield, UK Search for more papers by this author Mark J Dickman Mark J Dickman Department of Chemical and Biological Engineering, ChELSI Institute, University of Sheffield, Sheffield, UK Search for more papers by this author Rodolphe Barrangou Rodolphe Barrangou DuPont Nutrition and Health, Madison, WI, USA Search for more papers by this author Philippe Horvath Philippe Horvath DuPont Nutrition and Health, Dangé-Saint-Romain, France Search for more papers by this author Virginijus Siksnys Corresponding Author Virginijus Siksnys Department of Protein–DNA Interactions, Institute of Biotechnology, Vilnius University, Vilnius, Lithuania Search for more papers by this author Author Information Tomas Sinkunas1, Giedrius Gasiunas1, Sakharam P Waghmare2, Mark J Dickman2, Rodolphe Barrangou3, Philippe Horvath4 and Virginijus Siksnys 1 1Department of Protein–DNA Interactions, Institute of Biotechnology, Vilnius University, Vilnius, Lithuania 2Department of Chemical and Biological Engineering, ChELSI Institute, University of Sheffield, Sheffield, UK 3DuPont Nutrition and Health, Madison, WI, USA 4DuPont Nutrition and Health, Dangé-Saint-Romain, France *Corresponding author. Department of Protein–DNA Interactions, Institute of Biotechnology, Vilnius University, Graiciuno 8, Vilnius 02241, Lithuania. Tel.:+370 5 2602108; Fax:+370 5 2602116; E-mail: [email protected] The EMBO Journal (2013)32:385-394https://doi.org/10.1038/emboj.2012.352 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Clustered regularly interspaced short palindromic repeats (CRISPR)-encoded immunity in Type I systems relies on the Cascade (CRISPR-associated complex for antiviral defence) ribonucleoprotein complex, which triggers foreign DNA degradation by an accessory Cas3 protein. To establish the mechanism for adaptive immunity provided by the Streptococcus thermophilus CRISPR4-Cas (CRISPR-associated) system (St-CRISPR4-Cas), we isolated an effector complex (St-Cascade) containing 61-nucleotide CRISPR RNA (crRNA). We show that St-Cascade, guided by crRNA, binds in vitro to a matching proto-spacer if a proto-spacer adjacent motif (PAM) is present. Surprisingly, the PAM sequence determined from binding analysis is promiscuous and limited to a single nucleotide (A or T) immediately upstream (−1 position) of the proto-spacer. In the presence of a correct PAM, St-Cascade binding to the target DNA generates an R-loop that serves as a landing site for the Cas3 ATPase/nuclease. We show that Cas3 binding to the displaced strand in the R-loop triggers DNA cleavage, and if ATP is present, Cas3 further degrades DNA in a unidirectional manner. These findings establish a molecular basis for CRISPR immunity in St-CRISPR4-Cas and other Type I systems. Introduction Bacterial viruses (bacteriophages) provide a ubiquitous and often deadly threat to bacterial populations. To survive in hostile environments, bacteria have developed a multitude of antiviral defence systems (Sturino and Klaenhammer, 2006; Labrie et al, 2010). Clustered regularly interspaced short palindromic repeats (CRISPR) together with CRISPR-associated genes (cas) constitute an adaptive immune system, which provides acquired resistance against viruses and plasmids, in bacteria and archaea (Barrangou et al, 2007). The CRISPR-Cas system hijacks short fragments of invasive DNA, integrates them as spacers within the CRISPR array, and subsequently uses them as templates to generate specific small-interfering CRISPR RNA (crRNA) molecules that combine with Cas proteins into effector complexes that trigger degradation of matching foreign nucleic acids, thereby preventing their proliferation and propagation (Al-Attar et al, 2011; Bhaya et al, 2011; Terns and Terns, 2011; Wiedenheft et al, 2012). CRISPR-Cas systems have been categorized into three main types that differ by the structural organization and function(s) of nucleoprotein complexes involved in crRNA-mediated silencing of foreign nucleic acids (Makarova et al, 2011). In Type I systems (as exemplified by the CRISPR-Cas system of Escherichia coli K12), crRNAs are incorporated into a multisubunit ribonucleoprotein (RNP) complex called Cascade (CRISPR-associated complex for antiviral defence), which binds to matching invasive DNA and triggers degradation by an accessory Cas3 protein (Brouns et al, 2008; Sinkunas et al, 2011; Westra et al, 2012). In Type II systems (as exemplified by the CRISPR1-Cas and CRISPR3-Cas systems of Streptococcus thermophilus), CRISPR-mediated immunity solely relies on the signature Cas9 protein that associates with crRNA to form an effector complex, which specifically cleaves matching target double-stranded DNA (dsDNA) (Garneau et al, 2010; Deltcheva et al, 2011; Sapranauskas et al, 2011; Gasiunas et al, 2012; Jinek et al, 2012). In Type III systems (as exemplified by Sulfolobus solfataricus and Pyrococcus furiosus), Cas RAMP module (Cmr) in association with crRNA recognizes and cleaves RNA in vitro (Hale et al, 2012; Zhang et al, 2012), whereas the CRISPR-Cas system of Staphylococcus epidermidis targets DNA in vivo (Marraffini and Sontheimer, 2010). The S. thermophilus DGCC7710 model organism (St), for which CRISPR-Cas interference has been demonstrated against phages and plasmids, contains four distinct CRISPR-Cas systems (Horvath and Barrangou, 2010). Direct spacer acquisition and interference activities have been demonstrated for two distinct Type II systems, namely St-CRISPR1-Cas and St-CRISPR3-Cas (Barrangou et al, 2007; Deveau et al, 2008; Garneau et al, 2010; Sapranauskas et al, 2011; Gasiunas et al, 2012). However, until now, neither spacer acquisition nor interference activity has been reported for the St-CRISPR2-Cas or St-CRISPR4-Cas systems, which belong to Type III and Type I systems, respectively. Therefore, we investigated whether the St-CRISPR4-Cas system is functionally active and has the ability to provide immunity against invading DNA. The St-CRISPR4-Cas system of S. thermophilus DGCC7710 and E. coli CRISPR-Cas system are orthologous (Type I–E) and share genetic structural organization (Horvath and Barrangou, 2010; Sinkunas et al, 2011). In the St-CRISPR4-Cas system, five cas genes are arranged into a cluster (cse1-cse2-cas7-cas5-cas6e) (Figure 1A) analogous to the E. coli cas genes, suggesting that corresponding Cas proteins may assemble into a homologous St-Cascade complex. While in vivo functional activity has not been observed for the St-CRISPR4-Cas system, indirect in vitro evidence suggests that it may be active, at least at the interference step (Young et al, 2012). Indeed, we have previously shown that the Cas3 protein of the St-CRISPR4-Cas system is an active nuclease/helicase, which may play a key role in DNA degradation (Sinkunas et al, 2011). Figure 1.S. thermophilus CRISPR4-Cas crRNA and Cascade complex. (A) Schematic representation of the CRISPR4-Cas locus containing eight cas genes and twelve repeat-spacer units (conserved 28-bp palindromic repeats 5′-GTTTTTCCCGCACACGCGGGGGTGATCC-3′ are separated from each other by 33-bp spacers of variable sequence). St-Cascade genes homologous to the E. coli Cascade are underlined. Genes names according to Brouns et al (2008) and Makarova et al (2011) are indicated, respectively, above and below corresponding genes. (B) Coomassie blue-stained SDS-polyacrylamide gel of St-Cascade complex proteins isolated using the CasC-His6 protein as bait. (C) IP RP HPLC analysis of mature crRNA. (D) LC ESI-MS analysis of purified S. thermophilus crRNA. Inset shows an enhanced view of the 22-charge state. (E) Architecture of crRNA co-purifying with the St-Cascade protein complex.Source data for this figure is available on the online supplementary information page. Source Data for Figure 1 [embj2012352-sup-0001-SourceData-S1.pdf] Download figure Download PowerPoint Here, we report the isolation and biochemical characterization of the St-Cascade complex, which consists of CasABCDE proteins (Cse1, Cse2, Cas7, Cas5, and Cas6e, respectively) and a 61-nt crRNA. We further demonstrate that crRNA bound to the St-Cascade complex serves as the guide sequence, which specifically recognizes a matching sequence (proto-spacer) in the target DNA. We show that similarly to other Type I systems, St-Cascade binding to the proto-spacer in the invading DNA requires an additional DNA sequence element, a specific proto-spacer adjacent motif (PAM). However, in contrast with other CRISPR systems, the PAM for St-CRISPR4-Cas is promiscuous and limited to a single nucleotide. Furthermore, we show that St-Cascade and St-Cas3 form a functional effector complex, which cleaves target DNA in vitro. Results Cloning, expression, and isolation of St-Cascade In E. coli, CasABCDE proteins (Cse1, Cse2, Cas7, Cas5, and Cas6e, respectively) (Makarova et al, 2011), and crRNA form a Cascade complex (Ec-Cascade) (Brouns et al, 2008) which, together with Cas3, provide interference against invading foreign DNA. We tested the hypothesis that homologous S. thermophilus Cas proteins (Figure 1A) may assemble into a similar St-Cascade complex, and designed the following strategy for complex isolation. First, three compatible heterologous plasmids containing, respectively, a casABCDE cassette, the C-terminal His-tagged variant of casC (casC-His), and six copies of the repeat-spacer-1 unit (6 × SP1) of the S. thermophilus CRISPR4 region, were engineered. Next, all three plasmids were co-expressed in E. coli BL21 (DE3) strain and the St-Cascade complex was purified by subsequent Ni-chelating, size exclusion, and heparin affinity chromatography steps. Sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) analysis (Figure 1B) of the isolated complex revealed five bands that matched to individual Cas proteins, suggesting that CasABCDE proteins assemble into a St-Cascade complex similar to that of E. coli. The identity of all Cas proteins in St-Cascade was confirmed by mass spectrometry analysis (Supplementary Table S1). The stoichiometry of the protein complex was not directly determined; however, the band intensity in the SDS–PAGE (Figure 1B) in conjunction with the mass spectrometry analysis of the St-Cascade tryptic digest suggests that the CasC protein is the most abundant protein present in St-Cascade similar to the Ec-Cascade (Jore et al, 2011). Denaturing PAGE analysis revealed that RNA co-purifies with the St-Cascade complex (Supplementary Figure S1). Characterization of S. thermophilus CRISPR4-Cas crRNA Next, we used denaturing RNA chromatography in conjunction with electrospray ionization mass spectrometry (ESI-MS) to characterize the mature crRNAs isolated directly from the St-Cascade complex. Denaturing ion pair reverse phase chromatography was used to purify the crRNA directly from the St-Cascade complex (Dickman and Hornby, 2006; Waghmare et al, 2009). The RNA isolated from this complex consisted of a single mature crRNA with a retention time consistent with an approximate length of 60 nt (Figure 1C). Purified mature crRNA was further analysed using ESI-MS to obtain the accurate intact mass. A molecular weight of 19 482 Da was obtained (Figure 1D). In addition, ESI-MS/MS was used to analyse the oligoribonucleotide fragments generated from RNase T1 and RNase A digestion of the mature crRNA (Supplementary Figures S2 and S3). In conjunction with the intact mass analysis and denaturing PAGE (Supplementary Figure S1), these indicate that processing of St-CRISPR4-Cas crRNAs is similar to that of E. coli CRISPR-Cas crRNAs, generating a 61-nt crRNA (consisting of a 7-nt 5′ handle, a 33-nt spacer, and a 21-nt 3′ handle) with 5′-OH and 3′-Pi (MW 19 481.5 Da) (Figure 1E). Further verification of the 3′-Pi termini was obtained upon acid treatment of the crRNA where no change in mass was observed using ESI-MS. PAM sequence analysis of the S. thermophilus CRISPR4-Cas system The PAM located in the vicinity of a proto-spacer is absolutely required for silencing of invading DNA by Type I and Type II CRISPR-Cas systems (Deveau et al, 2008; Horvath et al, 2008; Sapranauskas et al, 2011; Semenova et al, 2011). In the E. coli Type I-E system, the PAM corresponds to the 5′-AWG-3′ sequence located immediately upstream of a proto-spacer (Mojica et al, 2009) and is essential for Ec-Cascade binding and subsequent DNA interference (Semenova et al, 2011). On the other hand, experimental analysis of CRISPR repeat boundaries in E. coli suggests a dinucleotide 5′-AW-3′ as PAM (Goren et al, 2012). To determine the putative PAM sequence of the CRISPR4-Cas system, we analysed all currently available CRISPR4 spacer sequences found in S. thermophilus strains. A CRISPR4 locus is present in DGCC7710 (Horvath and Barrangou, 2010) and three other strains from the DuPont culture collection. In DGCC7710, the CRISPR4 locus contains 12 unique spacers, and 26 more unique spacers were identified in the three other CRISPR4-positive strains. Sequence similarity searches, both in public and proprietary sequence databases, showed that most (26 out of 38) of these CRISPR4 spacer sequences have matches (proto-spacers) in S. thermophilus phage sequences. Only perfect matches (100% identity over the complete spacer sequence) between spacer and proto-spacer were considered, providing a set of 106 matching sequences. The sequences located immediately upstream and downstream of these proto-spacers were examined for the presence of a possible PAM. After removal of redundant alleles, a Weblogo representation (Crooks et al, 2004) was used to depict sequence conservation over a 15-nt segment of 28 (upstream) and 21 (downstream) unique sequences (Figure 2A). A 2-base pair (bp) conserved motif 5′-AA-3′ could be identified immediately upstream of the proto-spacers. Figure 2.PAM-dependent St-Cascade binding. (A) Predicted PAM for the St-CRISPR4-Cas system. Weblogo representation (Crooks et al, 2004) of 15-nt sequences found immediately upstream (top) and downstream (bottom) of phage proto-spacers that match known CRISPR4 spacers. A conserved, 2-nt PAM (5′-AA-3′) is located immediately upstream of the proto-spacers. (B) A schematic representation of a putative R-loop structure resulting from the St-Cascade binding to the 73-bp oligodeoxynucleotide. Nucleotides NN at −1 and −2 positions of predicted PAM were varied. In the R-loop structure, a target strand bound to the crRNA is engaged into a heteroduplex while the non-target strand is displaced as a single-stranded DNA. (C) PAM sequence dependence of a proto-spacer-1 binding by St-Cascade. Bar diagram shows dissociation constant Kd values obtained by EMSA. Error bars represent standard deviations of average Kd value determined in three separate experiments. Oligoduplex containing a non-matching proto-spacer-3 sequence was used as a non-specific DNA control. Download figure Download PowerPoint PAM is required for St-Cascade binding to the proto-spacer To determine whether the predicted PAM sequence is important for proto-spacer recognition, we analysed St-Cascade binding to a set of synthetic 73-bp oligoduplexes containing the spacer-1 sequence and variable nucleotides at positions −2 and −1 (Figure 2B). Oligoduplexes were radiolabelled at the 5′-end of the target strand, and the St-Cascade binding affinity was evaluated by electrophoretic mobility shift assay (EMSA). Binding analysis revealed that oligoduplexes fall into three categories with regards to St-Cascade binding. Oligoduplexes containing N(−2)A(−1) nucleotides in the predicted PAM display high binding affinity with Kd ∼0.2 nM, oligoduplexes containing N(−2)T(−1) nucleotides show binding with Kd <10 nM, while all other oligoduplexes except A(−2)G(−1) bind with the same affinity as the non-specific oligoduplex containing spacer-3 instead of spacer-1. Thus, these results suggest that a single nucleotide PAM, A or T (W) at the −1 position upstream of the proto-spacer is required for the St-CRISPR4-Cas system. The G and C nucleotides are not tolerated at this position except for the A(−2)G(−1) dinucleotide (Figure 2C; Supplementary Table S2). To test whether a non-conserved nucleotide at the −3 position in the vicinity of the predicted PAM is important for spacer recognition, we analysed St-Cascade binding to a set of oligoduplexes containing a conserved A(−2)A(−1) dinucleotide and any nucleotide at the −3 position (SP1-TAA, SP1-AAA, SP1-GAA, and SP1-CAA, respectively) (Supplementary Figure S4A). EMSA analysis revealed that St-Cascade bound all oligoduplexes with a variable N(−3) nt with the same affinity (Supplementary Figure S4B), confirming that the −3 position is not important for St-Cascade binding. In Type I CRISPR systems, as exemplified by E. coli and P. aeruginosa, target recognition is governed by the crRNA seed sequence located at the 5′-end of the spacer region (Semenova et al, 2011; Wiedenheft et al, 2011) and results in the formation of an R-loop where the target strand of the proto-spacer is engaged into a heteroduplex, while the non-target strand is displaced as single-stranded DNA (ssDNA). To demonstrate the formation of the R-loop upon St-Cascade binding to a proto-spacer, we used the P1 nuclease that specifically cleaves ssDNA regions (Jore et al, 2011; Supplementary Figure S5). In the oligoduplexes SP1-AA and SP1-AG that contain correct PAMs, the non-target strand is susceptible to endonuclease P1 cleavage, while the target strand is resistant to P1 nuclease treatment. On the other hand, in the oligoduplex SP1-CC, which lacks a correct PAM, or in the oligoduplex SP3-AA which contains a PAM but lacks a matching proto-spacer sequence, both DNA strands were resistant to nuclease P1 cleavage. Thus, nuclease P1 assay confirms that an R loop is formed only when both the correct PAM and a matching proto-spacer sequence are present in the oligoduplex. St-Cascade binding to the proto-spacer triggers St-Cas3 ATPase activity St-Cas3 is a metal-dependent nuclease that possesses a ssDNA stimulated ATPase activity coupled to unwinding of DNA/DNA and RNA/DNA duplexes (Sinkunas et al, 2011). St-Cascade complex binding to the proto-spacer creates an R-loop (Figure 2B) where a non-target strand is displaced as ssDNA and may function as a docking site for Cas3. We used a colorimetric assay to monitor St-Cas3 ATPase activity in the presence of St-Cascade and DNA. The St-Cas3 protein was mixed with the St-Cascade complex and pUC19 plasmid variants that either contain or lack proto-spacer-1 in the context of the correct or mutated PAM (Supplementary Table S3), and ATPase reactions were initiated by addition of ATP and Mg2+ ions. The assay revealed that ATPase activity of St-Cas3 is triggered only in the presence of the pSP1-AA plasmid, containing a matching proto-spacer-1 and a correct PAM (Figure 3A). Plasmids lacking proto-spacer-1 and PAM or containing a different proto-spacer (pSP3-AA) or incorrect CC PAM (pSP1-CC) did not stimulate St-Cas3 ATPase activity in the presence of St-Cascade complex. No ATPase activity was detected for the St-Cas3 ATPase-deficient mutant D452A with an impaired Walker B motif (Sinkunas et al, 2011). Taken together, these results suggest a link between the ATPase activity and Cas3 docking on ssDNA formed upon Cascade binding to the matching proto-spacer flanked by a correct PAM. Figure 3.ATPase and nuclease activities of St-Cas3 induced by St-Cascade binding to the proto-spacer dsDNA. (A) ATP hydrolysis rates. Malachite green assay was used to measure ATP hydrolysis through the detection of free phosphate liberated from ATP. Reaction rate constant k values (1/min) were calculated from linear slopes of time courses of phosphate liberation per St-Cas3 amount added. ATPase reactions were conducted at 37°C in the ATPase reaction buffer supplemented with 3 nM supercoiled plasmids, 12 nM St-Cascade and 300 nM St-Cas3 or the ATPase-deficient mutant D452A. Error bars indicate the ±standard deviation for the rate constant k value determined in three separate experiments. (B) dsDNA degradation requires St-Cas3, St-Cascade, and ATP. Nuclease reactions were conducted at 37°C for 10 min in a Nuclease buffer supplemented with 5 nM pSP1-AA and indicated amounts of St-Cas3 and St-Cascade. (C) PAM and a proto-spacer are essential for DNA degradation. Nuclease reactions were conducted at 37°C for indicated time intervals in Nuclease buffer supplemented with 100 nM St-Cas3, 20 nM St-Cascade, and 5 nM of respective supercoiled plasmids.Source data for this figure is available on the online supplementary information page. Source Data for Figure 3CD [embj2012352-sup-0002-SourceData-S2.pdf] Download figure Download PowerPoint St-Cascade binding to the proto-spacer triggers St-Cas3-mediated plasmid degradation Cas3 of S. thermophilus is a metal-dependent nuclease that degrades ssDNA using an active site located in the N-terminal HD domain (Sinkunas et al, 2011). dsDNA is resistant to St-Cas3 cleavage (Sinkunas et al, 2011). Consistent with published data on the Thermus thermophilus Cas3 protein (Mulepati and Bailey, 2011), St-Cas3 degraded ss M13mp18 DNA more rapidly in the presence of Ni2+ compared to Mg2+ ions (Supplementary Figure S6A). Since the ATPase activity of St-Cas3 is not supported by Ni2+ ions (Sinkunas et al, 2011), a mixture of Mg2+ and Ni2+ ions that supports both ATPase/helicase and nuclease activities of St-Cas3 was used in further experiments (Supplementary Figure S6B). To determine whether St-Cas3 docking on the ssDNA formed upon St-Cascade binding triggers nuclease activity, we analysed Cas3-mediated cleavage of plasmid DNA. pSP1-AA plasmid was pre-incubated with St-Cascade and an ATP solution containing Mg2+ and Ni2+ ions, followed by addition of St-Cas3. Under these conditions, the pSP1-AA plasmid was degraded in a St-Cascade and St-Cas3 concentration- and time-dependent manner (Figure 3B and C). On the other hand, the pUC19 plasmid, or plasmids containing a non-matching proto-spacer (pSP3-AA) or a defective PAM (pSP1-CC), were resistant to St-Cas3 cleavage (Figure 3C). ATP hydrolysis was required for pSP1-AA plasmid degradation. In the absence of ATP, the supercoiled pSP1-AA plasmid was converted into a nicked form but not degraded (Figure 3B). The identical cleavage pattern was observed for the St-Cas3 ATPase-deficient mutant D452A in the presence of ATP (Figure 3C). In contrast, the D227A replacement in the nuclease active site (Sinkunas et al, 2011) abolished DNA cleavage activity. Taken together, these data suggest that both ATPase/helicase and nuclease activities of St-Cas3 are required for pSP1-AA plasmid degradation in the presence of St-Cascade. It has been recently reported that Ec-Cascade preferentially binds a negatively supercoiled DNA and that a linear or nicked plasmid is not degraded by Ec-Cascade-Cas3 (Westra et al, 2012). To find out whether DNA supercoiling affects DNA cleavage rate by the St-Cascade-Cas3 system, we monitored degradation rates of supercoiled (pSP1-AA) and linearized (pSP1-AA-BamHI) DNA forms by St-Cascade-Cas3 (Supplementary Figure S12). In contrast to the E. coli CRISPR system, St-Cascade-Cas3 degraded both supercoiled and linear DNA substrates at similar rates (kSC=0.54±0.06/min and klinear=0.43±0.01/min, respectively). PAM is required for St-Cas3-mediated plasmid degradation DNA binding studies revealed that St-Cascade binding to a matching proto-spacer sequence requires a correct PAM sequence (Figure 2C). To check whether plasmid DNA cleavage in the in vitro reconstituted interference system follows the same dependence on PAM, we engineered plasmid substrates containing all possible combinations of base pairs at the −2 and −1 positions relative to the proto-spacer (within the predicted PAM), and monitored St-Cas3-mediated cleavage in the presence of St-Cascade and ATP (Supplementary Figure S7). Consistent with previous binding assays, we found that plasmids containing A(−1), T(−1), or A(−2)G(−1) nucleotides upstream of proto-spacer-1 were efficiently degraded, while plasmids with B(−2)G(−1) (where B=T or C or G) or C(−1) sequences were resistant to cleavage. Altogether, DNA binding and cleavage experiments demonstrate that proto-spacer recognition by St-Cascade is PAM dependent, and that subsequent R-loop formation triggers dsDNA degradation by St-Cas3. St-Cas3 cleaves DNA within the proto-spacer and upstream of the PAM To map St-Cas3 cleavage sites, oligoduplexes SP1-AA, SP1-CC, and SP3-AA were 33P-5′-end-labelled either on the target or non-target strand, and St-Cas3-induced cleavage was assessed on each strand of each duplex in the absence or presence of ATP. In the absence of ATP, only a non-target DNA strand of the SP1-AA oligoduplex is cut in the proto-spacer region, while the target DNA strand is resistant to cleavage (Figure 4A and C). In contrast, in the presence of ATP, both target and non-target strands of the SP1-AA oligoduplex are cleaved at multiple positions (Figure 4B). The non-target strand is extensively cut within the proto-spacer and upstream of the PAM at the 5′-end proximal region. The target strand is extensively cleaved within the proto-spacer with minor cuts occurring at both the 5′- and 3′-proximal termini (Figure 4D). Consistent with plasmid DNA cleavage data, no St-Cas3-mediated cleavage was observed for the oligoduplex lacking a proto-spacer (SP3-AA) or with a mutated PAM sequence (SP1-CC), neither in the presence or absence of ATP. Furthermore, the nuclease-deficient mutant D227A did not cleave the SP1-AA oligoduplex. In contrast, ATPase-deficient D452A mutant cleaved only the non-target strand, in both the absence and presence of ATP (Figure 4). The cleavage pattern of the SP1-AA oligoduplex explains why the nicked DNA form is a major product during plasmid DNA cleavage by the D452 mutant or wild-type (WT) St-Cas3 in the absence of ATP. Interestingly, almost all cleavage sites are located at the 3′-end of pyrimidine (T and C) bases (Supplementary Figure S8). Preference for pyrimidine bases is also characteristic for the St-Cas3 cleavage of single-stranded oligodeoxynucleotides (Supplementary Figure S9). The non-target strand cleavage at the 5′-end proximal side of the oligoduplex suggests subsequent St-Cas3 translocation in the 3′→5′ direction, followed by DNA degradation. Figure 4.St-Cas3 cleavage of St-Cascade bound to target dsDNA. Oligoduplexes 33P-labelled in either the non-target or target strand were pre-incubated with St-Cascade without (A) or with (B) ATP and reaction products analysed in denaturating polyacrylamide gels and mapped on the SP1-AA oligoduplex sequence (C, D), respectively. Cleavage reactions were conducted at 37°C in a Nuclease buffer containing 0 mM (A) or 2 mM (B) ATP, 8 nM St-Cascade and 100 nM (B) or 500 nM (A) St-Cas3 or D227A and D452A mutants supplemented with 2 nM SP1-AA, SP1-CC, or SP3-AA 33P-labelled oligoduplexes. Solid lines designate proto-spacer boundaries. Arrows indicate cleavage positions, height of the arrow correlates with a relative amount of cleavage product after 10 min incubation.Source data for this figure is available on the online supplementary information page. Source Data for Figure 4 [embj2012352-sup-0003-SourceData-S3.pdf] Download figure Download PowerPoint DNA degradation by Cas3 in the St-CRISPR4-Cas system is unidirectional To determine whether the St-Cas3-mediated DNA cleavage is directional, we linearized pSP1-AA plasmid using four different restriction endonucleases (XapI, BamHI, PdmI, AlwNI) to generate a set of linear dsDNA}, number={3}, journal={The EMBO Journal}, publisher={Wiley-Blackwell}, author={Sinkunas, Tomas and Gasiunas, Giedrius and Waghmare, Sakharam P and Dickman, Mark J and Barrangou, Rodolphe and Horvath, Philippe and Siksnys, Virginijus}, year={2013}, month={Jan}, pages={385–394} }
 @article{briner_barrangou_2014, title={Lactobacillus buchneri Genotyping on the Basis of Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) Locus Diversity}, volume={80}, ISSN={["1098-5336"]}, DOI={10.1128/aem.03015-13}, abstractNote={ABSTRACT
          
            Clustered regularly interspaced short palindromic repeats (CRISPR) in combination with associated sequences (
            cas
            ) constitute the CRISPR-Cas immune system, which uptakes DNA from invasive genetic elements as novel “spacers” that provide a genetic record of immunization events. We investigated the potential of CRISPR-based genotyping of
            Lactobacillus buchneri
            , a species relevant for commercial silage, bioethanol, and vegetable fermentations. Upon investigating the occurrence and diversity of CRISPR-Cas systems in
            Lactobacillus buchneri
            genomes, we observed a ubiquitous occurrence of CRISPR arrays containing a 36-nucleotide (nt) type II-A CRISPR locus adjacent to four
            cas
            genes, including the universal
            cas1
            and
            cas2
            genes and the type II signature gene
            cas9
            . Comparative analysis of CRISPR spacer content in 26
            L. buchneri
            pickle fermentation isolates associated with spoilage revealed 10 unique locus genotypes that contained between 9 and 29 variable spacers. We observed a set of conserved spacers at the ancestral end, reflecting a common origin, as well as leader-end polymorphisms, reflecting recent divergence. Some of these spacers showed perfect identity with phage sequences, and many spacers showed homology to
            Lactobacillus
            plasmid sequences. Following a comparative analysis of sequences immediately flanking protospacers that matched CRISPR spacers, we identified a novel putative protospacer-adjacent motif (PAM), 5′-AAAA-3′. Overall, these findings suggest that type II-A CRISPR-Cas systems are valuable for genotyping of
            L. buchneri
            .
          }, number={3}, journal={APPLIED AND ENVIRONMENTAL MICROBIOLOGY}, publisher={American Society for Microbiology}, author={Briner, Alexandra E. and Barrangou, Rodolphe}, year={2014}, month={Feb}, pages={994–1001} }
 @article{timme_pettengill_allard_strain_barrangou_wehnes_van kessel_karns_musser_brown_2013, title={Phylogenetic Diversity of the Enteric Pathogen Salmonella enterica subsp enterica Inferred from Genome-Wide Reference-Free SNP Characters}, volume={5}, ISSN={["1759-6653"]}, DOI={10.1093/gbe/evt159}, abstractNote={The enteric pathogen Salmonella enterica is one of the leading causes of foodborne illness in the world. The species is extremely diverse, containing more than 2,500 named serovars that are designated for their unique antigen characters and pathogenicity profiles—some are known to be virulent pathogens, while others are not. Questions regarding the evolution of pathogenicity, significance of antigen characters, diversity of clustered regularly interspaced short palindromic repeat (CRISPR) loci, among others, will remain elusive until a strong evolutionary framework is established. We present the first large-scale S. enterica subsp. enterica phylogeny inferred from a new reference-free k-mer approach of gathering single nucleotide polymorphisms (SNPs) from whole genomes. The phylogeny of 156 isolates representing 78 serovars (102 were newly sequenced) reveals two major lineages, each with many strongly supported sublineages. One of these lineages is the S. Typhi group; well nested within the phylogeny. Lineage-through-time analyses suggest there have been two instances of accelerated rates of diversification within the subspecies. We also found that antigen characters and CRISPR loci reveal different evolutionary patterns than that of the phylogeny, suggesting that a horizontal gene transfer or possibly a shared environmental acquisition might have influenced the present character distribution. Our study also shows the ability to extract reference-free SNPs from a large set of genomes and then to use these SNPs for phylogenetic reconstruction. This automated, annotation-free approach is an important step forward for bacterial disease tracking and in efficiently elucidating the evolutionary history of highly clonal organisms.}, number={11}, journal={GENOME BIOLOGY AND EVOLUTION}, publisher={Oxford University Press (OUP)}, author={Timme, Ruth E. and Pettengill, James B. and Allard, Marc W. and Strain, Errol and Barrangou, Rodolphe and Wehnes, Chris and Van Kessel, JoAnn S. and Karns, Jeffrey S. and Musser, Steven M. and Brown, Eric W.}, year={2013}, pages={2109–2123} }
 @article{horvath_barrangou_2013, title={RNA-guided genome editing à la carte}, volume={23}, DOI={10.1038/cr.2013.39}, abstractNote={Two recent papers in Science illustrate how the prokaryotic CRISPR-Cas immune system machinery, which typically targets invasive genetic elements such as viruses and plasmids, can be converted into a sophisticated molecular tool for next-generation human genome editing. The versatile Cas9 RNA-guided endonuclease can be readily reprogrammed using customizable small RNAs for sequence-specific single- or double-stranded DNA cleavage.}, number={6}, journal={Cell Research}, publisher={Springer Nature}, author={Horvath, Philippe and Barrangou, Rodolphe}, year={2013}, month={Mar}, pages={733–734} }
 @misc{abou hachem_andersen_barrangou_moller_fredslund_majumder_ejby_lahtinen_jacobsen_lo leggio_et al._2013, title={Recent insight into oligosaccharide uptake and metabolism in probiotic bacteria}, volume={31}, ISSN={["1029-2446"]}, DOI={10.3109/10242422.2013.828048}, abstractNote={Abstract In recent years, a plethora of studies have demonstrated the paramount physiological importance of the gut microbiota on various aspects of human health and development. Particular focus has been set on probiotic members of this community, the best studied of which are assigned into the Lactobacillus and Bifidobacterium genera. Effects such as pathogen exclusion, alleviation of inflammation and allergies, colon cancer, and other bowel disorders are attributed to the activity of probiotic bacteria, which selectively ferment prebiotics comprising mainly non-digestible oligosaccharides. Thus, glycan metabolism is an important attribute of probiotic action and a factor influencing the composition of the gut microbiota. In the quest to understand the molecular mechanism of this selectivity for certain glycans, we have explored the routes of uptake and utilization of a variety of oligosaccharides differing in size, composition, and glycosidic linkages. A combination of “omics” technologies bioinformatics, enzymology and protein characterization proved fruitful in elucidating the protein transport and catabolic machinery conferring the utilization of glucosides, galactosides, and xylosides in the two clinically validated probiotic strains Lactobacillus acidophilus NCFM and Bifidobacterium animalis subsp. lactis Bl-04. Importantly, we have been able to identify and in some cases validate the specificity of several transport systems, which are otherwise poorly annotated. Further, we have demonstrated for the first time that non-naturally occurring tri- and tetra-saccharides are internalized and efficiently utilized by probiotic bacteria in some cases better than well-established natural prebiotics. Selected highlights of these data are presented, emphasising the importance and the diversity of oligosaccharide transport in probiotic bacteria.}, number={4}, journal={BIOCATALYSIS AND BIOTRANSFORMATION}, publisher={Informa UK Limited}, author={Abou Hachem, Maher and Andersen, Joakim M. and Barrangou, Rodolphe and Moller, Marie S. and Fredslund, Folmer and Majumder, Avishek and Ejby, Morten and Lahtinen, Sampo J. and Jacobsen, Susanne and Lo Leggio, Leila and et al.}, year={2013}, month={Aug}, pages={226–235} }
 @article{paez-espino_morovic_sun_thomas_ueda_stahl_barrangou_banfield_2013, title={Strong bias in the bacterial CRISPR elements that confer immunity to phage}, volume={4}, DOI={10.1038/ncomms2440}, abstractNote={Clustered regularly interspaced short palindromic repeats (CRISPR)–Cas systems provide adaptive immunity against phage via spacer-encoded CRISPR RNAs that are complementary to invasive nucleic acids. Here, we challenge Streptococcus thermophilus with a bacteriophage, and used PCR-based metagenomics to monitor phage-derived spacers daily for 15 days in two experiments. Spacers that target the host chromosome are infrequent and strongly selected against, suggesting autoimmunity is lethal. In experiments that recover over half a million spacers, we observe early dominance by a few spacer sub-populations and rapid oscillations in sub-population abundances. In two CRISPR systems and in replicate experiments, a few spacers account for the majority of spacer sequences. Nearly all phage locations targeted by the acquired spacers have a proto-spacer adjacent motif (PAM), indicating PAMs are involved in spacer acquisition. We detect a strong and reproducible bias in the phage genome locations from which spacers derive. This may reflect selection for specific spacers based on location and effectiveness. Bacterial CRISPR–Cas systems provide adaptive immunity against phage by transcribing interfering RNA from phage DNA inserted into the bacterial genome. Using deep-sequencing, the authors detect a bias in the phage genome locations sampled, suggestive of selection.}, journal={Nature Communications}, publisher={Springer Nature}, author={Paez-Espino, David and Morovic, Wesley and Sun, Christine L. and Thomas, Brian C. and Ueda, Ken-ichi and Stahl, Buffy and Barrangou, Rodolphe and Banfield, Jillian F.}, year={2013}, month={Feb}, pages={1430} }
 @article{shariat_kirchner_sandt_trees_barrangou_dudley_2013, title={Subtyping of Salmonella enterica Serovar Newport Outbreak Isolates by CRISPR-MVLST and Determination of the Relationship between CRISPR-MVLST and PFGE Results}, volume={51}, ISSN={["0095-1137"]}, DOI={10.1128/jcm.00608-13}, abstractNote={ABSTRACTSalmonella entericasubsp.entericaserovar Newport (S. Newport) is the third most prevalent cause of food-borne salmonellosis. Rapid, efficient, and accurate methods for identification are required to track specific strains ofS. Newport during outbreaks. By exploiting the hypervariable nature of virulence genes and clustered regularly interspaced short palindromic repeats (CRISPRs), we previously developed a sequence-based subtyping approach, designated CRISPR–multi-virulence-locus sequence typing (CRISPR-MVLST). To demonstrate the applicability of this approach, we analyzed a broad set ofS. Newport isolates collected over a 5-year period by using CRISPR-MVLST and pulsed-field gel electrophoresis (PFGE). Among 84 isolates, we defined 38S. Newport sequence types (NSTs), all of which were novel compared to our previous analyses, and 62 different PFGE patterns. Our data suggest that both subtyping approaches have high discriminatory abilities (>0.95) with a potential for clustering cases with common exposures. Importantly, we found that isolates from closely related NSTs were often similar by PFGE profile as well, further corroborating the applicability of CRISPR-MVLST. In the first full application of CRISPR-MVLST, we analyzed isolates from a recentS. Newport outbreak. In this blinded study, we confirmed the utility of CRISPR-MVLST and were able to distinguish the 10 outbreak isolates, as defined by PFGE and epidemiological data, from a collection of 20S. Newport isolates. Together, our data show that CRISPR-MVLST could be a complementary approach to PFGE subtyping forS. Newport.}, number={7}, journal={JOURNAL OF CLINICAL MICROBIOLOGY}, publisher={American Society for Microbiology}, author={Shariat, Nikki and Kirchner, Margaret K. and Sandt, Carol H. and Trees, Eija and Barrangou, Rodolphe and Dudley, Edward G.}, year={2013}, month={Jul}, pages={2328–2336} }
 @article{yin_jensen_bai_debroy_barrangou_dudley_2013, title={The Evolutionary Divergence of Shiga Toxin-Producing Escherichia coli Is Reflected in Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) Spacer Composition}, volume={79}, DOI={10.1128/aem.00950-13}, abstractNote={ABSTRACT
          
            The Shiga toxin-producing
            Escherichia coli
            (STEC) strains, including those of O157:H7 and the “big six” serogroups (i.e., serogroups O26, O45, O103, O111, O121, and O145), are a group of pathogens designated food adulterants in the United States. The relatively conserved nature of
            c
            lustered
            r
            egularly
            i
            nterspaced
            s
            hort
            p
            alindromic
            r
            epeats (CRISPRs) in phylogenetically related
            E. coli
            strains makes them potential subtyping markers for STEC detection, and a quantitative PCR (qPCR)-based assay was previously developed for O26:H11, O45:H2, O103:H2, O111:H8, O121:H19, O145:H28, and O157:H7 isolates. To better evaluate the sensitivity and specificity of this qPCR method, the CRISPR loci of 252 O157 and big-six STEC isolates were sequenced and analyzed along with 563 CRISPR1 and 624 CRISPR2 sequences available in GenBank. General conservation of spacer content and order was observed within each O157 and big-six serogroup, validating the qPCR method. Meanwhile, it was found that spacer deletion, the presence of an insertion sequence, and distinct alleles within a serogroup are sources of false-negative reactions. Conservation of CRISPR arrays among isolates expressing the same flagellar antigen, specifically, H7, H2, and H11, suggested that these isolates share an ancestor and provided an explanation for the false positives previously observed in the qPCR results. An analysis of spacer distribution across
            E. coli
            strains provided limited evidence for temporal spacer acquisition. Conversely, comparison of CRISPR sequences between strains along the stepwise evolution of O157:H7 from its O55:H7 ancestor revealed that, over this ∼7,000-year span, spacer deletion was the primary force generating CRISPR diversity.
          }, number={18}, journal={Applied and Environmental Microbiology}, publisher={American Society for Microbiology}, author={Yin, S. and Jensen, M. A. and Bai, J. and DebRoy, C. and Barrangou, R. and Dudley, E. G.}, year={2013}, month={Jul}, pages={5710–5720} }
 @article{levin_moineau_bushman_barrangou_2013, title={The Population and Evolutionary Dynamics of Phage and Bacteria with CRISPR–Mediated Immunity}, volume={9}, DOI={10.1371/journal.pgen.1003312}, abstractNote={Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), together with associated genes (cas), form the CRISPR–cas adaptive immune system, which can provide resistance to viruses and plasmids in bacteria and archaea. Here, we use mathematical models, population dynamic experiments, and DNA sequence analyses to investigate the host–phage interactions in a model CRISPR–cas system, Streptococcus thermophilus DGCC7710 and its virulent phage 2972. At the molecular level, the bacteriophage-immune mutant bacteria (BIMs) and CRISPR–escape mutant phage (CEMs) obtained in this study are consistent with those anticipated from an iterative model of this adaptive immune system: resistance by the addition of novel spacers and phage evasion of resistance by mutation in matching sequences or flanking motifs. While CRISPR BIMs were readily isolated and CEMs generated at high rates (frequencies in excess of 10−6), our population studies indicate that there is more to the dynamics of phage–host interactions and the establishment of a BIM–CEM arms race than predicted from existing assumptions about phage infection and CRISPR–cas immunity. Among the unanticipated observations are: (i) the invasion of phage into populations of BIMs resistant by the acquisition of one (but not two) spacers, (ii) the survival of sensitive bacteria despite the presence of high densities of phage, and (iii) the maintenance of phage-limited communities due to the failure of even two-spacer BIMs to become established in populations with wild-type bacteria and phage. We attribute (i) to incomplete resistance of single-spacer BIMs. Based on the results of additional modeling and experiments, we postulate that (ii) and (iii) can be attributed to the phage infection-associated production of enzymes or other compounds that induce phenotypic phage resistance in sensitive bacteria and kill resistant BIMs. We present evidence in support of these hypotheses and discuss the implications of these results for the ecology and (co)evolution of bacteria and phage.}, number={3}, journal={PLoS Genetics}, publisher={Public Library of Science (PLoS)}, author={Levin, Bruce R. and Moineau, Sylvain and Bushman, Mary and Barrangou, Rodolphe}, editor={Hughes, DiarmaidEditor}, year={2013}, month={Mar}, pages={e1003312} }
 @article{shariat_dimarzio_yin_dettinger_sandt_lute_barrangou_dudley_2013, title={The combination of CRISPR-MVLST and PFGE provides increased discriminatory power for differentiating human clinical isolates of Salmonella enterica subsp. enterica serovar Enteritidis}, volume={34}, DOI={10.1016/j.fm.2012.11.012}, abstractNote={Salmonella enterica subsp. enterica serovar Enteritidis (S. Enteritidis) is a major cause of foodborne salmonellosis. Rapid, efficient and accurate methods for identification are required to track specific strains of S. Enteritidis during outbreaks of human salmonellosis. By exploiting the hypervariable nature of virulence genes and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs), we previously developed a powerful sequence-based subtyping approach, designated CRISPR-MVLST. To substantiate the applicability of CRISPR-MVLST, we analyzed a broad set of S. Enteritidis isolates collected over a six-year period. Among 141 isolates we defined 22 Enteritidis Sequence Types (ESTs), the majority of which were novel. Notably, strains exhibiting the common PFGE pattern, JEGX01.0004 (characteristic of ∼40% of S. Enteritidis isolates in the United States), were separated into twelve distinct sequence types. Conversely, isolates of EST4, the most predominant EST we observed, comprised eight different PFGE patterns. Importantly, we showed that some genotypes that were previously associated with the food supply chain at the farm level have now been identified in clinical samples. CRISPR sequence data shows subtle but distinct differences among different alleles of S. Enteritidis, suggesting that evolution of these loci occurs vertically, as opposed to previously reported evolution by spacer acquisition in other bacteria.}, number={1}, journal={Food Microbiology}, publisher={Elsevier BV}, author={Shariat, Nikki and DiMarzio, Michael J. and Yin, Shuang and Dettinger, Lisa and Sandt, Carol H. and Lute, James R. and Barrangou, Rodolphe and Dudley, Edward G.}, year={2013}, month={May}, pages={164–173} }
 @article{andersen_barrangou_abou hachem_lahtinen_goh_svensson_klaenhammer_2013, title={Transcriptional analysis of oligosaccharide utilization by Bifidobacterium lactis Bl-04}, volume={14}, ISSN={["1471-2164"]}, DOI={10.1186/1471-2164-14-312}, abstractNote={Abstract
          
            Background
            Probiotic bifidobacteria in combination with prebiotic carbohydrates have documented positive effects on human health regarding gastrointestinal disorders and improved immunity, however the selective routes of uptake remain unknown for most candidate prebiotics. The differential transcriptomes of Bifidobacterium animalis subsp. lactis Bl-04, induced by 11 potential prebiotic oligosaccharides were analyzed to identify the genetic loci involved in the uptake and catabolism of α- and β-linked hexoses, and β-xylosides.
          
          
            Results
            The overall transcriptome was modulated dependent on the type of glycoside (galactosides, glucosides or xylosides) utilized. Carbohydrate transporters of the major facilitator superfamily (induced by gentiobiose and β-galacto-oligosaccharides (GOS)) and ATP-binding cassette (ABC) transporters (upregulated by cellobiose, GOS, isomaltose, maltotriose, melibiose, panose, raffinose, stachyose, xylobiose and β-xylo-oligosaccharides) were differentially upregulated, together with glycoside hydrolases from families 1, 2, 13, 36, 42, 43 and 77. Sequence analysis of the identified solute-binding proteins that determine the specificity of ABC transporters revealed similarities in the breadth and selectivity of prebiotic utilization by bifidobacteria.
          
          
            Conclusion
            This study identified the differential gene expression for utilization of potential prebiotics highlighting the extensive capabilities of Bifidobacterium lactis Bl-04 to utilize oligosaccharides. Results provide insights into the ability of this probiotic microbe to utilize indigestible carbohydrates in the human gastrointestinal tract.
          }, number={1}, journal={BMC GENOMICS}, publisher={Springer Nature}, author={Andersen, Joakim M. and Barrangou, Rodolphe and Abou Hachem, Maher and Lahtinen, Sampo J. and Goh, Yong Jun and Svensson, Birte and Klaenhammer, Todd R.}, year={2013}, month={May} }
 @article{karvelis_gasiunas_miksys_barrangou_horvath_siksnys_2013, title={crRNA and tracrRNA guide Cas9-mediated DNA interference in Streptococcus thermophilus}, volume={10}, ISSN={["1555-8584"]}, DOI={10.4161/rna.24203}, abstractNote={The Cas9-crRNA complex of the Streptococcus thermophilus DGCC7710 CRISPR3-Cas system functions as an RNA-guided endonuclease with crRNA-directed target sequence recognition and protein-mediated DNA cleavage. We show here that an additional RNA molecule, tracrRNA (trans-activating CRISPR RNA), co-purifies with the Cas9 protein isolated from the heterologous E. coli strain carrying the S. thermophilus DGCC7710 CRISPR3-Cas system. We provide experimental evidence that tracrRNA is required for Cas9-mediated DNA interference both in vitro and in vivo. We show that Cas9 specifically promotes duplex formation between the precursor crRNA (pre-crRNA) transcript and tracrRNA, in vitro. Furthermore, the housekeeping RNase III contributes to primary pre-crRNA-tracrRNA duplex cleavage for mature crRNA biogenesis. RNase III, however, is not required in the processing of a short pre-crRNA transcribed from a minimal CRISPR array containing a single spacer. Finally, we show that an in vitro-assembled ternary Cas9-crRNA-tracrRNA complex cleaves DNA. This study further specifies the molecular basis for crRNA-based re-programming of Cas9 to specifically cleave any target DNA sequence for precise genome surgery. The processes for crRNA maturation and effector complex assembly established here will contribute to the further development of the Cas9 re-programmable system for genome editing applications.}, number={5}, journal={RNA BIOLOGY}, publisher={Informa UK Limited}, author={Karvelis, Tautvydas and Gasiunas, Giedrius and Miksys, Algirdas and Barrangou, Rodolphe and Horvath, Philippe and Siksnys, Virginijus}, year={2013}, month={May}, pages={841–851} }
 @article{broadbent_neeno-eckwall_stahl_tandee_cai_morovic_horvath_heidenreich_perna_barrangou_et al._2012, title={Analysis of the Lactobacillus casei supragenome and its influence in species evolution and lifestyle adaptation}, volume={13}, DOI={10.1186/1471-2164-13-533}, abstractNote={The broad ecological distribution of L. casei makes it an insightful subject for research on genome evolution and lifestyle adaptation. To explore evolutionary mechanisms that determine genomic diversity of L. casei, we performed comparative analysis of 17 L. casei genomes representing strains collected from dairy, plant, and human sources. Differences in L. casei genome inventory revealed an open pan-genome comprised of 1,715 core and 4,220 accessory genes. Extrapolation of pan-genome data indicates L. casei has a supragenome approximately 3.2 times larger than the average genome of individual strains. Evidence suggests horizontal gene transfer from other bacterial species, particularly lactobacilli, has been important in adaptation of L. casei to new habitats and lifestyles, but evolution of dairy niche specialists also appears to involve gene decay. Genome diversity in L. casei has evolved through gene acquisition and decay. Acquisition of foreign genomic islands likely confers a fitness benefit in specific habitats, notably plant-associated niches. Loss of unnecessary ancestral traits in strains collected from bacterial-ripened cheeses supports the hypothesis that gene decay contributes to enhanced fitness in that niche. This study gives the first evidence for a L. casei supragenome and provides valuable insights into mechanisms for genome evolution and lifestyle adaptation of this ecologically flexible and industrially important lactic acid bacterium. Additionally, our data confirm the Distributed Genome Hypothesis extends to non-pathogenic, ecologically flexible species like L. casei.}, number={1}, journal={BMC Genomics}, publisher={Springer Nature}, author={Broadbent, Jeff R and Neeno-Eckwall, Eric C and Stahl, Buffy and Tandee, Kanokwan and Cai, Hui and Morovic, Wesley and Horvath, Philippe and Heidenreich, Jessie and Perna, Nicole T and Barrangou, Rodolphe and et al.}, year={2012}, pages={533} }
 @article{horvath_gasiunas_siksnys_barrangou_2012, title={Applications of the Versatile CRISPR-Cas Systems}, DOI={10.1007/978-3-662-45794-8_11}, journal={CRISPR-Cas Systems}, publisher={Springer Berlin Heidelberg}, author={Horvath, Philippe and Gasiunas, Giedrius and Siksnys, Virginijus and Barrangou, Rodolphe}, year={2012}, pages={267–286} }
 @article{horvath_gasiunas_siksnys_barrangou_2012, title={Applications of the Versatile CRISPR-Cas Systems}, DOI={10.1007/978-3-642-34657-6_11}, journal={CRISPR-Cas Systems}, publisher={Springer Berlin Heidelberg}, author={Horvath, Philippe and Gasiunas, Giedrius and Siksnys, Virginijus and Barrangou, Rodolphe}, year={2012}, pages={267–286} }
 @book{barrangou_oost_2013, title={CRISPR-Cas Systems}, DOI={10.1007/978-3-642-34657-6}, publisher={Springer Berlin Heidelberg}, year={2013} }
 @article{barrangou_horvath_2012, title={CRISPR: New Horizons in Phage Resistance and Strain Identification}, volume={3}, DOI={10.1146/annurev-food-022811-101134}, abstractNote={ Bacteria have been widely used as starter cultures in the food industry, notably for the fermentation of milk into dairy products such as cheese and yogurt. Lactic acid bacteria used in food manufacturing, such as lactobacilli, lactococci, streptococci, Leuconostoc, pediococci, and bifidobacteria, are selectively formulated based on functional characteristics that provide idiosyncratic flavor and texture attributes, as well as their ability to withstand processing and manufacturing conditions. Unfortunately, given frequent viral exposure in industrial environments, starter culture selection and development rely on defense systems that provide resistance against bacteriophage predation, including restriction-modification, abortive infection, and recently discovered CRISPRs (clustered regularly interspaced short palindromic repeats). CRISPRs, together with CRISPR-associated genes (cas), form the CRISPR/Cas immune system, which provides adaptive immunity against phages and invasive genetic elements. The immunization process is based on the incorporation of short DNA sequences from virulent phages into the CRISPR locus. Subsequently, CRISPR transcripts are processed into small interfering RNAs that guide a multifunctional protein complex to recognize and cleave matching foreign DNA. Hypervariable CRISPR loci provide insights into the phage and host population dynamics, and new avenues for enhanced phage resistance and genetic typing and tagging of industrial strains. }, number={1}, journal={Annual Review of Food Science and Technology}, publisher={Annual Reviews}, author={Barrangou, Rodolphe and Horvath, Philippe}, year={2012}, month={Apr}, pages={143–162} }
 @article{gasiunas_barrangou_horvath_siksnys_2012, title={Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria}, volume={109}, DOI={10.1073/pnas.1208507109}, abstractNote={
            Clustered, regularly interspaced, short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems provide adaptive immunity against viruses and plasmids in bacteria and archaea. The silencing of invading nucleic acids is executed by ribonucleoprotein complexes preloaded with small, interfering CRISPR RNAs (crRNAs) that act as guides for targeting and degradation of foreign nucleic acid. Here, we demonstrate that the Cas9–crRNA complex of the
            Streptococcus thermophilus
            CRISPR3/Cas system introduces in vitro a double-strand break at a specific site in DNA containing a sequence complementary to crRNA. DNA cleavage is executed by Cas9, which uses two distinct active sites, RuvC and HNH, to generate site-specific nicks on opposite DNA strands. Results demonstrate that the Cas9–crRNA complex functions as an RNA-guided endonuclease with RNA-directed target sequence recognition and protein-mediated DNA cleavage. These findings pave the way for engineering of universal programmable RNA-guided DNA endonucleases.
          }, number={39}, journal={Proceedings of the National Academy of Sciences}, publisher={Proceedings of the National Academy of Sciences}, author={Gasiunas, G. and Barrangou, R. and Horvath, P. and Siksnys, V.}, year={2012}, month={Sep}, pages={E2579–E2586} }
 @article{stahl_barrangou_2012, title={Complete Genome Sequences of Probiotic Strains Bifidobacterium animalis subsp. lactis B420 and Bi-07}, volume={194}, DOI={10.1128/jb.00766-12}, abstractNote={ABSTRACT
          
            We present the complete genomes of
            Bifidobacterium animalis
            subsp.
            lactis
            B420 and Bi-07. Comparative genomic analysis with the type strain DSMZ10140 revealed 40 to 55 single nucleotide polymorphisms (SNPs) and an indel in a clustered regularly interspaced short palindromic repeat (CRISPR) locus. These genetic differences provide a molecular basis for strain typing within the two main phylogenetic groups of this monomorphic species.
          }, number={15}, journal={Journal of Bacteriology}, publisher={American Society for Microbiology}, author={Stahl, B. and Barrangou, R.}, year={2012}, month={Jul}, pages={4131–4132} }
 @article{weinberger_sun_pluciński_denef_thomas_horvath_barrangou_gilmore_getz_banfield_2012, title={Persisting Viral Sequences Shape Microbial CRISPR-based Immunity}, volume={8}, DOI={10.1371/journal.pcbi.1002475}, abstractNote={Well-studied innate immune systems exist throughout bacteria and archaea, but a more recently discovered genomic locus may offer prokaryotes surprising immunological adaptability. Mediated by a cassette-like genomic locus termed Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), the microbial adaptive immune system differs from its eukaryotic immune analogues by incorporating new immunities unidirectionally. CRISPR thus stores genomically recoverable timelines of virus-host coevolution in natural organisms refractory to laboratory cultivation. Here we combined a population genetic mathematical model of CRISPR-virus coevolution with six years of metagenomic sequencing to link the recoverable genomic dynamics of CRISPR loci to the unknown population dynamics of virus and host in natural communities. Metagenomic reconstructions in an acid-mine drainage system document CRISPR loci conserving ancestral immune elements to the base-pair across thousands of microbial generations. This ‘trailer-end conservation’ occurs despite rapid viral mutation and despite rapid prokaryotic genomic deletion. The trailer-ends of many reconstructed CRISPR loci are also largely identical across a population. ‘Trailer-end clonality’ occurs despite predictions of host immunological diversity due to negative frequency dependent selection (kill the winner dynamics). Statistical clustering and model simulations explain this lack of diversity by capturing rapid selective sweeps by highly immune CRISPR lineages. Potentially explaining ‘trailer-end conservation,’ we record the first example of a viral bloom overwhelming a CRISPR system. The polyclonal viruses bloom even though they share sequences previously targeted by host CRISPR loci. Simulations show how increasing random genomic deletions in CRISPR loci purges immunological controls on long-lived viral sequences, allowing polyclonal viruses to bloom and depressing host fitness. Our results thus link documented patterns of genomic conservation in CRISPR loci to an evolutionary advantage against persistent viruses. By maintaining old immunities, selection may be tuning CRISPR-mediated immunity against viruses reemerging from lysogeny or migration.}, number={4}, journal={PLoS Computational Biology}, publisher={Public Library of Science (PLoS)}, author={Weinberger, Ariel D. and Sun, Christine L. and Pluciński, Mateusz M. and Denef, Vincent J. and Thomas, Brian C. and Horvath, Philippe and Barrangou, Rodolphe and Gilmore, Michael S. and Getz, Wayne M. and Banfield, Jillian F.}, editor={Mering, ChristianEditor}, year={2012}, month={Apr}, pages={e1002475} }
 @article{sun_barrangou_thomas_horvath_fremaux_banfield_2012, title={Phage mutations in response to CRISPR diversification in a bacterial population}, volume={15}, DOI={10.1111/j.1462-2920.2012.02879.x}, abstractNote={SummaryInteractions between bacteria and their coexisting phage populations impact evolution and can strongly influence biogeochemical processes in natural ecosystems. Periodically, mutation or migration results in exposure of a host to a phage to which it has no immunity; alternatively, a phage may be exposed to a host it cannot infect. To explore the processes by which coexisting, co‐evolving hosts and phage populations establish, we cultured Streptococcus thermophilus DGCC7710 with phage 2972 and tracked CRISPR (clustered regularly interspaced short palindromic repeats) diversification and host–phage co‐evolution in a population derived from a colony that acquired initial CRISPR‐encoded immunity. After 1 week of co‐culturing, the coexisting host–phage populations were metagenomically characterized using 454 FLX Titanium sequencing. The evolved genomes were compared with reference genomes to identify newly incorporated spacers in S. thermophilus DGCC7710 and recently acquired single‐nucleotide polymorphisms (SNPs) in phage 2972. Following phage exposure, acquisition of immune elements (spacers) led to a genetically diverse population with multiple subdominant strain lineages. Phage mutations that circumvented three early immunization events were localized in the proto‐spacer adjacent motif (PAM) or near the PAM end of the proto‐spacer, suggesting a strong selective advantage for the phage that mutated in this region. The sequential fixation or near fixation of these single mutations indicates selection events so severe that single phage genotypes ultimately gave rise to all surviving lineages and potentially carried traits unrelated to immunity to fixation.}, number={2}, journal={Environmental Microbiology}, publisher={Wiley-Blackwell}, author={Sun, Christine L. and Barrangou, Rodolphe and Thomas, Brian C. and Horvath, Philippe and Fremaux, Christophe and Banfield, Jillian F.}, year={2012}, month={Oct}, pages={463–470} }
 @article{young_dill_pan_hettich_banfield_shah_fremaux_horvath_barrangou_verberkmoes_2012, title={Phage-Induced Expression of CRISPR-Associated Proteins Is Revealed by Shotgun Proteomics in Streptococcus thermophilus}, volume={7}, DOI={10.1371/journal.pone.0038077}, abstractNote={The CRISPR/Cas system, comprised of clustered regularly interspaced short palindromic repeats along with their associated (Cas) proteins, protects bacteria and archaea from viral predation and invading nucleic acids. While the mechanism of action for this acquired immunity is currently under investigation, the response of Cas protein expression to phage infection has yet to be elucidated. In this study, we employed shotgun proteomics to measure the global proteome expression in a model system for studying the CRISPR/Cas response in S. thermophilus DGCC7710 infected with phage 2972. Host and viral proteins were simultaneously measured following inoculation at two different multiplicities of infection and across various time points using two-dimensional liquid chromatography tandem mass spectrometry. Thirty-seven out of forty predicted viral proteins were detected, including all proteins of the structural virome and viral effector proteins. In total, 1,013 of 2,079 predicted S. thermophilus proteins were detected, facilitating the monitoring of host protein synthesis changes in response to virus infection. Importantly, Cas proteins from all four CRISPR loci in the S. thermophilus DGCC7710 genome were detected, including loci previously thought to be inactive. Many Cas proteins were found to be constitutively expressed, but several demonstrated increased abundance following infection, including the signature Cas9 proteins from the CRISPR1 and CRISPR3 loci, which are key players in the interference phase of the CRISPR/Cas response. Altogether, these results provide novel insights into the proteomic response of S. thermophilus, specifically CRISPR-associated proteins, upon phage 2972 infection.}, number={5}, journal={PLoS ONE}, publisher={Public Library of Science (PLoS)}, author={Young, Jacque C. and Dill, Brian D. and Pan, Chongle and Hettich, Robert L. and Banfield, Jillian F. and Shah, Manesh and Fremaux, Christophe and Horvath, Philippe and Barrangou, Rodolphe and VerBerkmoes, Nathan C.}, editor={Bruggemann, HolgerEditor}, year={2012}, month={May}, pages={e38077} }
 @article{barrangou_2012, title={RNA-mediated programmable DNA cleavage}, volume={30}, DOI={10.1038/nbt.2357}, number={9}, journal={Nature Biotechnology}, publisher={Springer Nature}, author={Barrangou, Rodolphe}, year={2012}, month={Sep}, pages={836–838} }
 @article{andersen_barrangou_abou hachem_lahtinen_goh_svensson_klaenhammer_2012, title={Transcriptional Analysis of Prebiotic Uptake and Catabolism by Lactobacillus acidophilus NCFM}, volume={7}, ISSN={["1932-6203"]}, DOI={10.1371/journal.pone.0044409}, abstractNote={The human gastrointestinal tract can be positively modulated by dietary supplementation of probiotic bacteria in combination with prebiotic carbohydrates. Here differential transcriptomics and functional genomics were used to identify genes in Lactobacillus acidophilus NCFM involved in the uptake and catabolism of 11 potential prebiotic compounds consisting of α- and β- linked galactosides and glucosides. These oligosaccharides induced genes encoding phosphoenolpyruvate-dependent sugar phosphotransferase systems (PTS), galactoside pentose hexuronide (GPH) permease, and ATP-binding cassette (ABC) transporters. PTS systems were upregulated primarily by di- and tri-saccharides such as cellobiose, isomaltose, isomaltulose, panose and gentiobiose, while ABC transporters were upregulated by raffinose, Polydextrose, and stachyose. A single GPH transporter was induced by lactitol and galactooligosaccharides (GOS). The various transporters were associated with a number of glycoside hydrolases from families 1, 2, 4, 13, 32, 36, 42, and 65, involved in the catabolism of various α- and β-linked glucosides and galactosides. Further subfamily specialization was also observed for different PTS-associated GH1 6-phospho-β-glucosidases implicated in the catabolism of gentiobiose and cellobiose. These findings highlight the broad oligosaccharide metabolic repertoire of L. acidophilus NCFM and establish a platform for selection and screening of both probiotic bacteria and prebiotic compounds that may positively influence the gastrointestinal microbiota.}, number={9}, journal={PLOS ONE}, publisher={Public Library of Science (PLoS)}, author={Andersen, Joakim Mark and Barrangou, Rodolphe and Abou Hachem, Maher and Lahtinen, Sampo J. and Goh, Yong-Jun and Svensson, Birte and Klaenhammer, Todd R.}, editor={Gibas, CynthiaEditor}, year={2012}, month={Sep} }
 @article{bhaya_davison_barrangou_2011, title={CRISPR-Cas Systems in Bacteria and Archaea: Versatile Small RNAs for Adaptive Defense and Regulation}, volume={45}, DOI={10.1146/annurev-genet-110410-132430}, abstractNote={Bacteria and archaea have evolved defense and regulatory mechanisms to cope with various environmental stressors, including virus attack. This arsenal has been expanded by the recent discovery of the versatile CRISPR-Cas system, which has two novel features. First, the host can specifically incorporate short sequences from invading genetic elements (virus or plasmid) into a region of its genome that is distinguished by clustered regularly interspaced short palindromic repeats (CRISPRs). Second, when these sequences are transcribed and precisely processed into small RNAs, they guide a multifunctional protein complex (Cas proteins) to recognize and cleave incoming foreign genetic material. This adaptive immunity system, which uses a library of small noncoding RNAs as a potent weapon against fast-evolving viruses, is also used as a regulatory system by the host. Exciting breakthroughs in understanding the mechanisms of the CRISPR-Cas system and its potential for biotechnological applications and understanding evolutionary dynamics are discussed.}, number={1}, journal={Annual Review of Genetics}, publisher={Annual Reviews}, author={Bhaya, Devaki and Davison, Michelle and Barrangou, Rodolphe}, year={2011}, pages={273–297} }
 @article{sinkunas_gasiunas_fremaux_barrangou_horvath_siksnys_2011, title={Cas3 is a single-stranded DNA nuclease and ATP-dependent helicase in the CRISPR/Cas immune system}, volume={30}, DOI={10.1038/emboj.2011.41}, abstractNote={Article22 February 2011free access Cas3 is a single-stranded DNA nuclease and ATP-dependent helicase in the CRISPR/Cas immune system Tomas Sinkunas Tomas Sinkunas Department of Protein–DNA Interactions, Institute of Biotechnology, Vilnius University, Vilnius, Lithuania Search for more papers by this author Giedrius Gasiunas Giedrius Gasiunas Department of Protein–DNA Interactions, Institute of Biotechnology, Vilnius University, Vilnius, Lithuania Search for more papers by this author Christophe Fremaux Christophe Fremaux Danisco France SAS, Dangé-Saint-Romain, France Search for more papers by this author Rodolphe Barrangou Rodolphe Barrangou Danisco USA Inc., Madison, WI, USA Search for more papers by this author Philippe Horvath Philippe Horvath Danisco France SAS, Dangé-Saint-Romain, France Search for more papers by this author Virginijus Siksnys Corresponding Author Virginijus Siksnys Department of Protein–DNA Interactions, Institute of Biotechnology, Vilnius University, Vilnius, Lithuania Search for more papers by this author Tomas Sinkunas Tomas Sinkunas Department of Protein–DNA Interactions, Institute of Biotechnology, Vilnius University, Vilnius, Lithuania Search for more papers by this author Giedrius Gasiunas Giedrius Gasiunas Department of Protein–DNA Interactions, Institute of Biotechnology, Vilnius University, Vilnius, Lithuania Search for more papers by this author Christophe Fremaux Christophe Fremaux Danisco France SAS, Dangé-Saint-Romain, France Search for more papers by this author Rodolphe Barrangou Rodolphe Barrangou Danisco USA Inc., Madison, WI, USA Search for more papers by this author Philippe Horvath Philippe Horvath Danisco France SAS, Dangé-Saint-Romain, France Search for more papers by this author Virginijus Siksnys Corresponding Author Virginijus Siksnys Department of Protein–DNA Interactions, Institute of Biotechnology, Vilnius University, Vilnius, Lithuania Search for more papers by this author Author Information Tomas Sinkunas1, Giedrius Gasiunas1, Christophe Fremaux2, Rodolphe Barrangou3, Philippe Horvath2 and Virginijus Siksnys 1 1Department of Protein–DNA Interactions, Institute of Biotechnology, Vilnius University, Vilnius, Lithuania 2Danisco France SAS, Dangé-Saint-Romain, France 3Danisco USA Inc., Madison, WI, USA *Corresponding author. Department of Protein–DNA Interactions, Institute of Biotechnology, Vilnius University, Graiciuno 8, Vilnius 02241, Lithuania. Tel.: +370 5 2602108; Fax: +370 5 2602116; E-mail: [email protected] The EMBO Journal (2011)30:1335-1342https://doi.org/10.1038/emboj.2011.41 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Clustered regularly interspaced short palindromic repeat (CRISPR) is a recently discovered adaptive prokaryotic immune system that provides acquired immunity against foreign nucleic acids by utilizing small guide crRNAs (CRISPR RNAs) to interfere with invading viruses and plasmids. In Escherichia coli, Cas3 is essential for crRNA-guided interference with virus proliferation. Cas3 contains N-terminal HD phosphohydrolase and C-terminal Superfamily 2 (SF2) helicase domains. Here, we provide the first report of the cloning, expression, purification and in vitro functional analysis of the Cas3 protein of the Streptococcus thermophilus CRISPR4 (Ecoli subtype) system. Cas3 possesses a single-stranded DNA (ssDNA)-stimulated ATPase activity, which is coupled to unwinding of DNA/DNA and RNA/DNA duplexes. Cas3 also shows ATP-independent nuclease activity located in the HD domain with a preference for ssDNA substrates. To dissect the contribution of individual domains, Cas3 separation-of-function mutants (ATPase+/nuclease− and ATPase−/nuclease+) were obtained by site-directed mutagenesis. We propose that the Cas3 ATPase/helicase domain acts as a motor protein, which assists delivery of the nuclease activity to Cascade–crRNA complex targeting foreign DNA. Introduction Viruses have a major influence on all types of cellular life including eukaryotes, bacteria and archaea. To protect themselves against infection, prokaryotes have developed multiple defence barriers of various complexity, including prevention of adsorption, blocking of injection or degradation of the foreign nucleic acid (Sturino and Klaenhammer, 2006; Labrie et al, 2010). Recently, an adaptive microbial immune system, named clustered regularly interspaced short palindromic repeats (CRISPRs), has been identified that provides acquired immunity against viruses and plasmids (Barrangou et al, 2007). It consists of an array of short conserved DNA-repeat sequences that are interspaced by stretches of variable sequence called spacers, which generally originate from phage or plasmid DNA (Bolotin et al, 2005; Mojica et al, 2005). A set of cas (CRISPR-associated) genes is typically located in the vicinity to repeat-spacer array (Jansen et al, 2002; Makarova et al, 2006). CRISPR, in combination with the associated Cas proteins, forms the CRISPR/Cas systems, which are currently grouped into eight subtypes on the basis of clustering of cas genes (Haft et al, 2005; Makarova et al, 2006). It was shown in Streptococcus thermophilus that during natural generation of phage-resistant variants, bacteria commonly alter their CRISPR loci by polarized incorporation of novel spacers, which originate from the phages used in the challenge (Barrangou et al, 2007; Deveau et al, 2008; Horvath et al, 2008). Mechanistically, the function of the CRISPR system has been dissected into two distinct steps: (i) CRISPR adaptation (immunization), which includes the recognition of foreign DNA followed by its subsequent processing and integration into the host chromosomal CRISPR locus and (ii) interference (immunity), which includes transcription of the CRISPR locus and generation of crRNAs that serve as the guide sequences in the binding and/or degradation of the target nucleic acid (Sorek et al, 2008; Hale et al, 2009; van der Oost et al, 2009; Horvath and Barrangou, 2010; Karginov and Hannon, 2010). The molecular mechanism by which CRISPR provides resistance against foreign genetic elements is yet to be characterized. The first mechanistic details on the interference (immunity) step (Brouns et al, 2008) were obtained in experimental studies of the CRISPR/Cas system in Escherichia coli. E. coli K-12 strain possesses a single cluster of cas genes (Ecoli subtype) encoding eight proteins: three core proteins, Cas1, Cas2 and Cas3, and a subtype-specific five-protein set, Cse1–Cse2–Cse4–Cas5e–Cse3. The latter proteins are also referred to as CasABCDE and constitute a so-called Cascade complex. It was demonstrated (Brouns et al, 2008) in E. coli K-12 strain that the CRISPR region is transcribed into a long transcript (termed pre-crRNA), which is processed by the Cascade complex into small crRNAs, which contain a single spacer flanked on both sides by short nucleotide stretches originating from the repeat. These crRNA bound to the Cascade complex serve as the guide sequences that target foreign DNA and result in the CRISPR interference, presumably through the binding and/or degradation of the target nucleic acid. In E. coli, the Cascade complex alone is able to generate crRNA but requires Cas3 to achieve immunity against foreign DNA (Brouns et al, 2008). Hence, Cas3 appears to be a key protein of CRISPR system, which is necessary for crRNA-guided interference of virus proliferation. Moreover, cas3 is present in most CRISPR/Cas subtypes (except Nmeni and Mtube) (Haft et al, 2005; Makarova et al, 2006) but the mechanism by which Cas3 achieves its function is not yet known. In silico analysis predicts that cas3 encodes a polypeptide comprised of HD-type phosphohydrolase/nuclease and DExD/H-box helicase domains (Haft et al, 2005; Makarova et al, 2006); however, so far there is no experimental evidence to demonstrate its role in vitro. Detailed mechanistic and structural studies of Cas3 have been limited, in part, by the lack of a suitable model system for carrying out quantitative biochemical and structural studies in vitro. Due to its poor solubility and aggregation, the E. coli Cas3 protein can only be expressed in a very limited amount (Gasiunas and Siksnys, unpublished data). The HD-type nuclease subunit of the related Sulfolobus solfataricus Cas3 protein has recently been expressed in a soluble form as a fusion protein and demonstrated to catalyse hydrolysis of double-stranded (ds)DNA and RNA, although no experimental details are yet available on the activity of the putative helicase domain (Han and Krauss, 2009). In this paper, we provide the first biochemical characterization of the Cas3 protein identified in the CRISPR4 system of S. thermophilus DGCC7710. The latter CRISPR system belongs to the Ecoli subtype and comprises eight genes arranged similarly to the CRISPR system of E. coli (Horvath and Barrangou, 2010) (Figure 1A). We have cloned and expressed the cas3 gene of S. thermophilus DGCC7710 in E. coli ER2267, and purified the Cas3 protein to an apparent homogeneity. Here, we provide the first direct experimental evidence that Cas3 is a multifunctional protein possessing single-stranded DNA (ssDNA)-stimulated ATPase, ATP-dependent helicase and a metal-dependent single-stranded nuclease activities. Figure 1.(A) Schematic organization of the CRISPR4/Cas system of S. thermophilus DGCC7710 and the CRISPR/Cas system of E. coli K-12. Percentage of identical and similar (in parenthesis) residues between corresponding protein sequences that are connected by dashed lines. Conserved repeat sequences of each system are shown in the inserts. (B) Domain architecture of the S. thermophilus Cas3 protein. Domains identified by in silico analysis are shown as grey boxes. HD domain denotes HD-type phosphohydrolase/nuclease domain; DExD/H domain denotes DExD/H-box helicase domain; HelC dom denotes the C-terminal helicase domain. Conserved residues characteristic of the different domains and subject to alanine mutagenesis are indicated above the boxes. Location of the conserved helicase motifs are indicated by numbers I, II and VI. Download figure Download PowerPoint Results Expression and purification of Cas3 protein According to the genomic sequence of S. thermophilus DGCC7710, the cas3 gene of the CRISPR4/Cas system encodes a protein of 926 amino acids with a predicted molecular mass of ∼106 kDa. The sequence data have been submitted to the GenBank database under accession No. HQ453272. In silico analysis of Cas3 protein reveals a multidomain architecture (Figure 1B). The N-terminal part of Cas3 shows conserved residues characteristic of the HD family of metal-dependent phosphohydrolases (HD domain) (Aravind and Koonin, 1998), whereas the C-terminal part of Cas3 has motifs characteristic of the Superfamily 2 (SF2) helicases (Singleton et al, 2007). A BLAST search revealed that the Cas3 protein from S. thermophilus DGCC7710 shows ∼22% identity and ∼60% similarity at the amino-acid level to the Cas3 protein of E. coli K-12 (Figure 1). The cas3 gene from S. thermophilus DGCC7710 was cloned into the pBAD24-C(His)6 plasmid to yield a construct encoding a fusion protein containing a C-terminal 6-His-tag. The recombinant plasmid was expressed in the E. coli strain ER2267 and the Cas3 protein purified from the crude cell extracts. The purified Cas3 protein was ∼95% homogeneous as evaluated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and Coomassie Blue staining (Supplementary Figure S2). Typically, ∼12 mg of protein was obtained in a single run from 1 l of E. coli culture. Cas3 shows an ssDNA-stimulated ATPase activity The presence of the characteristic helicase motifs (I, II and VI) responsible for ATP binding and hydrolysis in the primary sequence of Cas3 predicts that the protein would be an ATPase. The ability of Cas3 to hydrolyze ATP was first examined by monitoring the hydrolysis of radioactively labelled [α32P]ATP (Figure 2A). In the presence of the Cas3 protein alone, only minimal ATPase activity was detected. However, the ATP hydrolysis rate increased significantly in the presence of M13 circular ssDNA. We further investigated the ATP hydrolysis by Cas3 in the presence of ssDNA, dsDNA and RNA by measuring the concentration of accumulated phosphate product using a colorimetric assay (Hyun et al, 2008). Data analysis revealed that ATPase activity of Cas3 increased significantly in the presence of ssDNA but was not stimulated by the dsDNA or by RNA (Figure 2B). The linear accumulation of inorganic phosphate as a function of time (representative trace shown in Figure 2C) allowed us to calculate the rate of ATP hydrolysis at 0.5 mM of ATP as ∼38 min−1. Taken together, these results indicate that Cas3 possesses an ssDNA-dependent ATPase activity. Figure 2.Cas3 ATPase activity. (A) Radioactive ATPase assay. ATPase reactions were conducted at 30°C in a reaction buffer containing 10 mM Tris–HCl (pH 7.5 at 25°C), 30 mM KCl, 5% (v/v) glycerol, 2 mM MgCl2, 0.1 mg/ml BSA, 0.5 mM ATP, 2.5 nM ssDNA (M13mp18) or dsDNA (supercoiled form of pUC57 plasmid), and 250 nM Cas3. Reaction mixtures were supplemented with [α32P]ATP (5 Ci/mmol), spotted onto a polyethyleneimine-cellulose thin-layer plate and separated by chromatography followed by phosphorimager visualization. (B) ATP hydrolysis dependence of nucleic acids. Malachite green assay was used to measure ATP hydrolysis through the detection of liberated-free phosphate from ATP. Reaction mixtures in the buffer described above contained varying amounts of ssDNA (M13mp18), dsDNA (supercoiled form of pUC57 plasmid) or 2223 nt RNA. (C) Time courses of ATP hydrolysis. Reaction mixtures contained 3 nM ssDNA (M13mp18). Malachite green assay was used to measure ATP hydrolysis through the detection of liberated-free phosphate from ATP. (D) ATP hydrolysis rates. Reaction rate constant k (min−1) calculated from slopes of times courses shown in (C). Download figure Download PowerPoint To test whether the conserved residues of the helicase domain are important for the Cas3 ATPase activity, we generated a set of alanine-substitution mutants. We mutated amino-acid residues Q290A (located in Q-motif (Tanner et al, 2003) important for ATP binding), K316A (located in motif I involved in ATP binding), D452A and E453A (located in motif II involved in Mg2+ coordination at the ATPase active site), R663A and R666A (located in motif VI (Tanner and Linder, 2001) involved in ATP binding). As illustrated in Figure 2D, the ATPase activity of the mutants decreased significantly, indicating the importance of the conserved amino-acid residues for the ATP binding/hydrolysis. Conversely, mutations in the N-terminal HD domain had no effect on the ATPase activity of Cas3. In order to test the nucleotide specificity of Cas3, we compared the catalytic activity using ribo- and deoxyribonucleotide cofactors (Supplementary Figure S3). Cas3 exhibited a strong preference towards ATP or dATP. We also found that GTP and dGTP could be hydrolyzed in the presence of ssDNA, but with specific activities that were approximately 10-fold lower than observed with ATP. No significant hydrolytic activity was observed with other nucleotides. Study of the divalent metal ion dependence of the Cas3 ATPase activity revealed that the fastest rate was observed with Mn2+, followed closely by Mg2+ or Ca2+. Cu2+ supported hydrolysis at a much lower rate. No significant ATPase activity above background was observed using Co2+, Zn2+ or Ni2+ (Supplementary Figure S4). Cas3 shows nuclease activity located in the HD domain The nuclease activity of Cas3 was analysed using circular M13mp18 ssDNA or pUC57 supercoiled double-stranded plasmid DNA (Figure 3). Cas3 degraded the M13mp18 ssDNA in a concentration- and time-dependent manner (Figure 3; Supplementary Figure S5). In contrast, virtually no hydrolysis of the dsDNA occurred during the 2-h incubation. Mg2+ ions but not ATP were required for the ssDNA hydrolysis. Indeed, ssDNA was degraded with similar rates both in the absence or in the presence of ATP (data not shown). Figure 3.Cas3 nuclease activity. (A) Degradation of the ssDNA and dsDNA. Various amounts of Cas3 were incubated in the presence of 4 nM of M13 ssDNA or pUC57 dsDNA at 37°C for 2 h in the presence (+) or absence (−) of 10 mM MgCl2 or 10 mM EDTA. (B) Effect of mutations on Cas3 nuclease activity. In all, 500 nM of protein was incubated in the presence of 4 nM of M13 ssDNA at 37°C for 2 h. Download figure Download PowerPoint Conserved amino-acid residues H27, H76, D77 and D276 located in the N-terminal HD-like domain (Aravind and Koonin, 1998) of Cas3 (Figure 1B; Supplementary Figure S1) were identified as being part of the putative active site responsible for divalent metal binding and ssDNA hydrolysis. Alanine replacement mutants D77A and D227A were constructed by site-directed mutagenesis, mutant proteins were purified and their ability to degrade DNA was analysed (Figure 3B). Experimental data indicate that while ssDNA was fully degraded in 2 h by the wild type (wt) Cas3, ssDNA hydrolysis was significantly reduced for D77A and D227A. Mutations in the helicase domain had much weaker effects on the associated nuclease activity (Figure 3B). Cas3 shows DNA unwinding activity An ssDNA-dependent ATPase activity of Cas3 suggests a possible translocase/helicase function. To analyse whether the purified Cas3 protein possesses true helicase activity (i.e. DNA strand separation), we determined its capacity to unwind duplex DNA substrates (Figure 4). Since many helicases require a stretch of ssDNA in order to load onto the substrate and since Cas3 ATPase activity is stimulated by ssDNA, the substrate was constructed by hybridizing a 20-nt oligodeoxynucleotide, labelled with 32P at its 5′-terminus, to a circular M13mp18 ssDNA that contained a complementary sequence (Matson, 1986) (Figure 4A). This substrate was incubated with Cas3 and the reaction products were separated on a polyacrylamide gel. The labelled oligonucleotide was then detected by autoradiography. The experimental results indicate (Figure 4B) that Cas3 possesses a DNA unwinding activity that is dependent upon the presence of both Mg2+ and ATP but is not supported in the presence of the non-hydrolysable ATP analogue 5′-adenylyl-β, γ-imidodiphosphate (AMP-PNP). Furthermore, the Cas3 nuclease-deficient mutant D77A unwound the DNA similarly to the wt protein while the D452A mutant, which has compromised ATPase activity, showed no unwinding activity. Figure 4.Cas3 helicase activity and polarity. (A). Schematic representation of duplex unwinding assay. (B, C) DNA–DNA and RNA–DNA duplex unwinding by Cas3. Cas3 displacement of a P32-labelled 20 nt oligodeoxynucleotide (B) or 22 oligoribonucleotide (C) annealed to an ssM13mp18 DNA is monitored in the polyacrylamide gel. Reactions were performed at 30°C for 1 h in the reaction buffer: 10 mM Tris–HCl (pH 7.5 at 25°C), 25 mM KCl, 15% (v/v) glycerol, 1 mM MgCl2, 0.1 mg/ml BSA, 2 mM ATP, 0.5 nM substrate and various amounts of protein. nA denotes the ATP analogue AMP-PNP. D452A and D77A are ATPase and nuclease domain mutants, respectively. (D) Cas3 polarity assay I. Cas3 displacement of 30 nt double-stranded fragments at the ends of the linear M13mp18 DNA. Partial duplex DNA was prepared by EheI cleavage of the labelled 60 nt oligodeoxynucleotide annealed to the M13pm18 DNA. Duplex regions are separated by a single-stranded region of few thousand nucleotides. Reaction mixture contained 500 nM of Cas3. Reactions were stopped at defined time intervals. (E) Cas3 polarity assay II. Cas3 displacement of the oligonucleotide-based 73 nt substrates containing 53 nt 3′- or 5′-overhangs. Reactions were performed as in Figure 4B and C except that with the oligonucleotide-based substrates 500 nM of Cas3, 1.5 nM of substrate and 250 nM trap DNA (unlabelled oligonucleotide) were used in the reaction. Download figure Download PowerPoint To test whether the Cas3 protein unwinds an RNA/DNA heteroduplex, a substrate was constructed by hybridizing a 22-nt oligoribonucleotide to M13mp18 ssDNA as mentioned above. The experimental results indicate (Figure 4C) that Cas3 displaces the 22-nt oligoribonucleotide in the presence of ATP and Mg2+ ions. Thus, Cas3 can be classified as both a DNA–DNA and DNA–RNA helicase. With partially duplex substrates, each DNA helicase is thought to bind first to an ssDNA region and then to approach and unwind duplex DNA in a particular direction. To determine the directionality of Cas3, we first prepared a pair of labelled helicase substrates (Figure 4D) that contained duplex DNA at both ends of a long linear ssDNA molecule. Since the substrates comprise duplex regions at both ends of a long linear molecule, Cas3 must first bind to the internal single-stranded regions of these substrates. If the enzyme subsequently moves into a 3′–5′ direction along the ssDNA segment, it would displace the 5′-labelled fragment from the substrate. In contrast, the 3′-labelled fragment would be displaced if the enzyme migrates in a 5′–3′ direction. Experimental data presented in Figure 4 indicate that Cas3 moves primarily 3′ to 5′ along the ssDNA segment. We also performed an alternative unwinding assay using radiolabelled DNA substrates of different structures. These 73 nt partial oligoduplexes contained 53 nt 3′- or 5′-single-stranded overhangs in addition to a 20-bp duplex region (Figure 4E). Cas3 could only unwind the substrates containing a 3′-overhang, confirming the 3′–5′ polarity seen above. The unwinding activity of Cas3 was observed in the presence of ATP and was not detected in the absence of ATP or in the presence of the non-hydrolyzable ATP analogue (AMP-PNP), suggesting that ATP hydrolysis is required for helicase function. Discussion cas3 is a core gene present in most of the CRISPR/Cas subtypes (except Nmeni and Mtube). The presence of the HD-type phosphohydrolase/nuclease domain led to the proposal that Cas3 might catalyse crRNA-guided cleavage of foreign nucleic acids (van der Oost et al, 2009). Since Cas3 also carries an apparent ATP/helicase domain, it was suggested (Marraffini and Sontheimer, 2010) that it could act also as a target DNA unwindase to enable protospacer hybridization with the crRNA. Here, we overproduced the Cas3 protein of a S. thermophilus CRISPR4/Cas system (Horvath and Barrangou, 2010), which belongs to the Ecoli subtype, purified Cas3 to ∼95% homogeneity, and performed functional characterization in vitro. Cas3 degrades ssDNA The Cas3 protein of S. thermophilus is arranged as a polypeptide comprised of an N-terminal HD-type phosphohydrolase/nuclease domain and a C-terminal SF2 helicase domain (Haft et al, 2005; Makarova et al, 2006). This architecture is characteristic of most Cas3 proteins but it is not absolutely conserved. For example, in the Cas3 protein of Apern subtype, the domains are found in swapped order, while in the Cas3 of Ypest, the HD-type phosphohydrolase/nuclease domain is found at the N-terminus of the cas2 gene (Brouns et al, 2008). In some cases, such as in S. solfataricus, the HD-type phosphohydrolase/nuclease and the DExD/H-box helicase domains are encoded as separate genes (Han et al, 2009). The HD-type phosphohydrolase/nuclease domain is found in a superfamily of enzymes with either a predicted or known phosphohydrolase activity (Aravind and Koonin, 1998). According to Prosite database, bacterial HD domains are found in combination of 49 different partner domains, which presumably modulate protein function. HD-domain-containing proteins appear to be involved in nucleic acid metabolism and signal transduction, as well as other unknown functions (Aravind and Koonin, 1998). For example, the HD domain of the E. coli tRNA nucleotidyltransferase exhibits 2,3-cyclic phosphodiesterase, 2-nucleotidase and phosphatase activities (Yakunin et al, 2004). The N-terminal domain of Cas3 contains a characteristic signature of the HD-type phosphohydrolase/nuclease domain (Figure 1B). The conserved residues (H…HD…D) predicted to be involved in the coordination of the divalent metal (Aravind and Koonin, 1998) are conserved in the N-terminal domain of S. thermophilus Cas3 and correspond to the residues H27, H76, D77 and D227. We show here that in the presence of magnesium ions, Cas3 has a nuclease activity that degrades ssDNA. It does not act on dsDNA (Figure 3). While Mg2+ ions are absolutely necessary for the nuclease activity, ATP is not. Mutation of the key metal-coordinating residues D77 and D227 compromised the ability of Cas3 to degrade ssDNA, but did not affect the ATPase activity, consistent with in silico predictions. The ssDNA-degrading activity of the S. thermophilus Cas3 protein is in contrast to the apparent dsDNA and dsRNA cleavage activity of the standalone HD phosphohydrolase/nuclease of S. solfataricus (Han and Krauss, 2009) with only minor degradation of ssDNA or ssRNA observed. It is not clear whether this represents a true difference between the CRISPR systems or whether the activity of the HD domain is altered when the helicase domain is present (even though helicase activity is not required). Cas3 unwinds DNA in the presence of ATP The C-terminal fragment of Cas3 protein carries signature motifs characteristic of the SF2 helicases of the DExD/H subgroup (Singleton et al, 2007). Helicases use ATP to unwind and translocate nucleic acids or remodel nucleic acids or nucleic acid–protein complexes (Cordin et al, 2006; Singleton et al, 2007; Fairman-Williams et al, 2010). We show here that the Cas3 protein exhibits ssDNA-dependent ATPase activity, which absolutely requires Mg2+ ions. Nine conserved domains Q, I, Ia, Ib and II–VI have been identified in the SF2 group of DExD/H-type helicases (Cordin et al, 2006; Singleton et al, 2007; Fairman-Williams et al, 2010). The highest level of sequence conservation in the SF1 and SF2 helicase families is seen in the residues that coordinate binding and hydrolysis of the triphosphate (motifs I, II and VI). Mutations of the amino-acid residues Q290, K316, D452, E453, R663 and R666 in the conserved motifs I, II and VI, abolished or significantly compromised ATP hydrolysis. However, none of the helicase mutants showed any change in the ssDNA-degrading activity. Among different deoxy- and ribonucleotides tested, only ATP or dATP were hydrolysed significantly (Supplementary Figure S3). The maximum rate of ATP hydrolysis was ∼38 molecules per minute (Figure 2D). This value is also close to the value reported for the bacterial XPB helicase (Biswas et al, 2009) and NS3 helicase from hepatitis C virus (Kyono et al, 2003). The inefficient ATP hydrolysis suggests that Cas3 alone is not very processive or that it is an intrinsically slow ATPase involved, for example, in the local remodelling of protein–crRNA complex (see below). In parallel to the ATPase activity, Cas3 protein is able to unwind oligonucleotide–M13 DNA complex. The unwinding activity of Cas3 is dependent on the protein concentration and presence of ATP and Mg2+ ions. Using a linear M13 molecule with a large internal region of ssDNA where a helicase can assemble, and two 32P-labelled duplex regions of different lengths, we show that Cas3 helicase has 3′ → 5′ directionality. It is likely that Cas3 functions as DNA translocase but translocation activity still has to be demonstrated directly. Cas3 contains a long C-terminal extension that follows the conserved helicase domains of the SF2 superfamily. Terminal domains of helicases have been demonstrated to direct recruitment of partner proteins/complexes, to promote interactions with other proteins, or to facilitate recognition of specific nucleic acid regions (Karow and Klostermeier, 2010). One cannot exclude that the C-terminal domain of Cas3 performs similar functions. Putative role of Cas3 in the CRISPR mechanism Taken together, our biochemical studies show that Cas3 protein combines in a single protein both ssDNA nuclease and ATPase/helicase activities. How can these activities be reconciled with the CRISPR mechanism? It was previously shown that in E. coli at least, a so-called Cascade complex consisting of the five protein set Cse1–Cse2–Cse4–Cas5e–Cse3 performs processing of a pre-crRNA into mature crRNAs, which remain associated with Cascade (Brouns et al, 2008). Hence, Cas3 is not required in the crRNA generation step and is probably involved in the CRISPR adaptation and/or interference steps. Indeed, in vivo studies demonstrate that both Cas3 and Cascade–crRNA complexes are required for the interference step (Brouns et al, 2008). We suggest that ATP-dependent DNA helicase activity of Cas3 can contribute both to the protospacer sequence recognition by the Cascade–crRNA complex and DNA cleavage (Figure 5). It is possible that Cascade–crRNA complex interactions with the target DNA promote strand separation and create ssDNA regions for the Cas3 binding, which further unwinds DNA strands promoting crRNA hybridization to the complementary protospacer DNA. Indeed, Cas3 ATPase activity, which is necessary for the strand displacement, is stimulated by ssDNA but not by dsDNA (Figure 2). Invasion of the crRNA into the DNA duplex should result in the formation of the R-loop structure (Camps and Loeb, 2005) and the ATP-dependent Cas3 helicase activity may promote the formation of such loops. On the other hand, in the R-loop structure, a single-stranded antisense DNA s}, number={7}, journal={The EMBO Journal}, publisher={Wiley-Blackwell}, author={Sinkunas, Tomas and Gasiunas, Giedrius and Fremaux, Christophe and Barrangou, Rodolphe and Horvath, Philippe and Siksnys, Virginijus}, year={2011}, month={Feb}, pages={1335–1342} }
 @article{makarova_haft_barrangou_brouns_charpentier_horvath_moineau_mojica_wolf_yakunin_et al._2011, title={Evolution and classification of the CRISPR–Cas systems}, volume={9}, DOI={10.1038/nrmicro2577}, abstractNote={The CRISPR–Cas (clustered regularly interspaced short palindromic repeats–CRISPR-associated proteins) systems are immunity systems that are present in many bacteria and archaea. Here, Koonin and colleagues present a new classification of these systems and introduce a new nomenclature of the genes in the CRISPR–casloci that better reflects the relationships between the proteins. The CRISPR–Cas (clustered regularly interspaced short palindromic repeats–CRISPR-associated proteins) modules are adaptive immunity systems that are present in many archaea and bacteria. These defence systems are encoded by operons that have an extraordinarily diverse architecture and a high rate of evolution for both the cas genes and the unique spacer content. Here, we provide an updated analysis of the evolutionary relationships between CRISPR–Cas systems and Cas proteins. Three major types of CRISPR–Cas system are delineated, with a further division into several subtypes and a few chimeric variants. Given the complexity of the genomic architectures and the extremely dynamic evolution of the CRISPR–Cas systems, a unified classification of these systems should be based on multiple criteria. Accordingly, we propose a 'polythetic' classification that integrates the phylogenies of the most common cas genes, the sequence and organization of the CRISPR repeats and the architecture of the CRISPR–cas loci.}, number={6}, journal={Nature Reviews Microbiology}, publisher={Springer Nature}, author={Makarova, Kira S. and Haft, Daniel H. and Barrangou, Rodolphe and Brouns, Stan J. J. and Charpentier, Emmanuelle and Horvath, Philippe and Moineau, Sylvain and Mojica, Francisco J. M. and Wolf, Yuri I. and Yakunin, Alexander F. and et al.}, year={2011}, month={May}, pages={467–477} }
 @article{barrangou_lahtinen_ibrahim_ouwehand_2011, title={Genus Lactobacillus}, DOI={10.1201/b11503-6}, abstractNote={Contents 2.1 Introduction ......................................................................................................................18 2.2 Comparative Genomics of LAB ........................................................................................18}, journal={Lactic Acid Bacteria}, publisher={CRC Press}, author={Barrangou, Rodolphe and Lahtinen, Sampo and Ibrahim, Fandi and Ouwehand, Arthur}, year={2011}, pages={77–91} }
 @article{barrangou_horvath_2011, title={Lactic Acid Bacteria Defenses Against Phages}, DOI={10.1007/978-0-387-92771-8_19}, journal={Stress Responses of Lactic Acid Bacteria}, publisher={Springer US}, author={Barrangou, Rodolphe and Horvath, Philippe}, year={2011}, pages={459–478} }
 @article{liu_barrangou_gerner-smidt_ribot_knabel_dudley_2011, title={Novel Virulence Gene and Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) Multilocus Sequence Typing Scheme for Subtyping of the Major Serovars of Salmonella enterica subsp. enterica}, volume={77}, DOI={10.1128/aem.02625-10}, abstractNote={ABSTRACT
          
            Salmonella enterica
            subsp.
            enterica
            is the leading cause of bacterial food-borne disease in the United States. Molecular subtyping methods are powerful tools for tracking the farm-to-fork spread of food-borne pathogens during outbreaks. In order to develop a novel multilocus sequence typing (MLST) scheme for subtyping the major serovars of
            S. enterica
            subsp.
            enterica
            , the virulence genes
            sseL
            and
            fimH
            and clustered regularly interspaced short palindromic repeat (CRISPR) loci were sequenced from 171 clinical isolates from nine
            Salmonella
            serovars,
            Salmonella
            serovars Typhimurium, Enteritidis, Newport, Heidelberg, Javiana, I 4,[5],12:i:−, Montevideo, Muenchen, and Saintpaul. The MLST scheme using only virulence genes was congruent with serotyping and identified epidemic clones but could not differentiate outbreaks. The addition of CRISPR sequences dramatically improved discriminatory power by differentiating individual outbreak strains/clones. Of particular note, the present MLST scheme provided better discrimination of
            Salmonella
            serovar Enteritidis strains than pulsed-field gel electrophoresis (PFGE). This method showed high epidemiologic concordance for all serovars screened except for
            Salmonella
            serovar Muenchen. In conclusion, the novel MLST scheme described in the present study accurately differentiated outbreak strains/clones of the major serovars of
            Salmonella
            , and therefore, it shows promise for subtyping this important food-borne pathogen during investigations of outbreaks.
          }, number={6}, journal={Applied and Environmental Microbiology}, publisher={American Society for Microbiology}, author={Liu, F. and Barrangou, R. and Gerner-Smidt, P. and Ribot, E. M. and Knabel, S. J. and Dudley, E. G.}, year={2011}, month={Jan}, pages={1946–1956} }
 @article{loquasto_barrangou_dudley_roberts_2011, title={Short communication: The complete genome sequence of Bifidobacterium animalis subspecies animalis ATCC 25527T and comparative analysis of growth in milk with B. animalis subspecies lactis DSM 10140T}, volume={94}, DOI={10.3168/jds.2011-4499}, abstractNote={<h2>Abstract</h2> The objective of this work was to sequence the genome of <i>Bifidobacterium animalis</i> ssp. <i>animalis</i> ATCC 25527<sup>T</sup>, the subspecies most closely related to <i>B. animalis</i> ssp. <i>lactis</i>, some strains of which are widely added to dairy foods as probiotics. The complete 1,932,963-bp genome was determined by a combination of 454-shotgun sequencing and PCR gap closing, and the completed assembly was verified by comparison with a <i>Kpn</i>I optical map. Comparative analysis of the <i>B. animalis</i> ssp. <i>animalis</i> ATCC 25527<sup>T</sup> and <i>B. animalis</i> ssp. <i>lactis</i> DSM 10140<sup>T</sup> genomes revealed high degrees of synteny and sequence homology. Comparative genomic analysis revealed 156 and 182 genes that were unique to and absent in the <i>B. animalis</i> ssp. <i>animalis</i> genome, respectively. Among these was a set of unique clustered regularly interspaced short palindromic repeats (CRISPR)-associated genes and a novel CRISPR locus containing 30 spacers in the genome of <i>B. animalis</i> ssp. <i>animalis</i>. Although previous researchers have suggested that one of the defining phenotypic differences between <i>B. animalis</i> ssp. <i>animalis</i> and <i>B. animalis</i> ssp. <i>lactis</i> is the ability of the latter to grow in milk and milk-based media, the differential gene content did not provide insights to explain these differences. Furthermore, growth and acid production in milk and milk-based media did not differ significantly between <i>B. animalis</i> ssp. <i>lactis</i> (DSM 10140<sup>T</sup> and Bl04) and <i>B. animalis</i> ssp. <i>animalis</i> (ATCC 25527<sup>T</sup>). Growth of these strains in supplemented milk suggested that growth was limited by a lack of available low-molecular-weight nitrogen in the 3 strains examined.}, number={12}, journal={Journal of Dairy Science}, publisher={American Dairy Science Association}, author={Loquasto, J.R. and Barrangou, R. and Dudley, E.G. and Roberts, R.F.}, year={2011}, pages={5864–5870} }
 @article{liu_kariyawasam_jayarao_barrangou_gerner-smidt_ribot_knabel_dudley_2011, title={Subtyping Salmonella enterica Serovar Enteritidis Isolates from Different Sources by Using Sequence Typing Based on Virulence Genes and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs)}, volume={77}, DOI={10.1128/aem.00468-11}, abstractNote={ABSTRACT
          
            Salmonella enterica
            subsp.
            enterica
            serovar Enteritidis is a major cause of food-borne salmonellosis in the United States. Two major food vehicles for
            S
            . Enteritidis are contaminated eggs and chicken meat. Improved subtyping methods are needed to accurately track specific strains of
            S
            . Enteritidis related to human salmonellosis throughout the chicken and egg food system. A sequence typing scheme based on virulence genes (
            fimH
            and
            sseL
            ) and clustered regularly interspaced short palindromic repeats (CRISPRs)—CRISPR-including multi-virulence-locus sequence typing (designated CRISPR-MVLST)—was used to characterize 35 human clinical isolates, 46 chicken isolates, 24 egg isolates, and 63 hen house environment isolates of
            S
            . Enteritidis. A total of 27 sequence types (STs) were identified among the 167 isolates. CRISPR-MVLST identified three persistent and predominate STs circulating among U.S. human clinical isolates and chicken, egg, and hen house environmental isolates in Pennsylvania, and an ST that was found only in eggs and humans. It also identified a potential environment-specific sequence type. Moreover, cluster analysis based on
            fimH
            and
            sseL
            identified a number of clusters, of which several were found in more than one outbreak, as well as 11 singletons. Further research is needed to determine if CRISPR-MVLST might help identify the ecological origins of
            S
            . Enteritidis strains that contaminate chickens and eggs.
          }, number={13}, journal={Applied and Environmental Microbiology}, publisher={American Society for Microbiology}, author={Liu, F. and Kariyawasam, S. and Jayarao, B. M. and Barrangou, R. and Gerner-Smidt, P. and Ribot, E. M. and Knabel, S. J. and Dudley, E. G.}, year={2011}, month={May}, pages={4520–4526} }
 @article{sapranauskas_gasiunas_fremaux_barrangou_horvath_siksnys_2011, title={The Streptococcus thermophilus CRISPR/Cas system provides immunity in Escherichia coli}, volume={39}, DOI={10.1093/nar/gkr606}, abstractNote={The CRISPR/Cas adaptive immune system provides resistance against phages and plasmids in Archaea and Bacteria. CRISPR loci integrate short DNA sequences from invading genetic elements that provide small RNA-mediated interference in subsequent exposure to matching nucleic acids. In Streptococcus thermophilus, it was previously shown that the CRISPR1/Cas system can provide adaptive immunity against phages and plasmids by integrating novel spacers following exposure to these foreign genetic elements that subsequently direct the specific cleavage of invasive homologous DNA sequences. Here, we show that the S. thermophilus CRISPR3/Cas system can be transferred into Escherichia coli and provide heterologous protection against plasmid transformation and phage infection. We show that interference is sequence-specific, and that mutations in the vicinity or within the proto-spacer adjacent motif (PAM) allow plasmids to escape CRISPR-encoded immunity. We also establish that cas9 is the sole cas gene necessary for CRISPR-encoded interference. Furthermore, mutation analysis revealed that interference relies on the Cas9 McrA/HNH- and RuvC/RNaseH-motifs. Altogether, our results show that active CRISPR/Cas systems can be transferred across distant genera and provide heterologous interference against invasive nucleic acids. This can be leveraged to develop strains more robust against phage attack, and safer organisms less likely to uptake and disseminate plasmid-encoded undesirable genetic elements.}, number={21}, journal={Nucleic Acids Research}, publisher={Oxford University Press (OUP)}, author={Sapranauskas, Rimantas and Gasiunas, Giedrius and Fremaux, Christophe and Barrangou, Rodolphe and Horvath, Philippe and Siksnys, Virginijus}, year={2011}, month={Aug}, pages={9275–9282} }
 @article{andersen_barrangou_abou hachem_lahtinen_goh_svensson_klaenhammer_2011, title={Transcriptional and functional analysis of galactooligosaccharide uptake by lacS in Lactobacillus acidophilus}, volume={108}, ISSN={["0027-8424"]}, DOI={10.1073/pnas.1114152108}, abstractNote={Probiotic microbes rely on their ability to survive in the gastrointestinal tract, adhere to mucosal surfaces, and metabolize available energy sources from dietary compounds, including prebiotics. Genome sequencing projects have proposed models for understanding prebiotic catabolism, but mechanisms remain to be elucidated for many prebiotic substrates. Although β-galactooligosaccharides (GOS) are documented prebiotic compounds, little is known about their utilization by lactobacilli. This study aimed to identify genetic loci inLactobacillus acidophilusNCFM responsible for the transport and catabolism of GOS. Whole-genome oligonucleotide microarrays were used to survey the differential global transcriptome during logarithmic growth ofL. acidophilusNCFM using GOS or glucose as a sole source of carbohydrate. Within the 16.6-kbpgal-lacgene cluster,lacS, a galactoside-pentose-hexuronide permease-encoding gene, was up-regulated 5.1-fold in the presence of GOS. In addition, two β-galactosidases, LacA and LacLM, and enzymes in the Leloir pathway were also encoded by genes within this locus and up-regulated by GOS stimulation. Generation of alacS-deficient mutant enabled phenotypic confirmation of the functional LacS permease not only for the utilization of lactose and GOS but also lactitol, suggesting a prominent role of LacS in the metabolism of a broad range of prebiotic β-galactosides, known to selectively modulate the beneficial gut microbiota.}, number={43}, journal={PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA}, publisher={Proceedings of the National Academy of Sciences}, author={Andersen, Joakim M. and Barrangou, Rodolphe and Abou Hachem, Maher and Lahtinen, Sampo and Goh, Yong Jun and Svensson, Birte and Klaenhammer, Todd R.}, year={2011}, month={Oct}, pages={17785–17790} }
 @article{putaala_barrangou_leyer_ouwehand_hansen_romero_rautonen_2010, title={Analysis of the human intestinal epithelial cell transcriptional response toLactobacillus acidophilus, Lactobacillus salivarius, Bifidobacterium lactisandEscherichia coli}, volume={1}, DOI={10.3920/bm2010.0003}, abstractNote={The complex microbial population residing in the human gastrointestinal tract consists of commensal, potential pathogenic and beneficial species, which are probably perceived differently by the host and consequently could be expected to trigger specific transcriptional responses. Here, we provide a comparative analysis of the global in vitro transcriptional response of human intestinal epithelial cells to Lactobacillus acidophilus NCFM™, Lactobacillus salivarius Ls-33, Bifidobacterium animalis subsp. lactis 420, and enterohaemorrhagic Escherichia coli O157:H7 (EHEC). Interestingly, L. salivarius Ls-33 DCE-induced changes were overall more similar to those of B. lactis 420 than to L. acidophilus NCFM™, which is consistent with previously observed in vivo immunomodulation properties. In the gene ontology and pathway analyses both specific and unspecific changes were observed. Common to all was the regulation of apoptosis and adipogenesis, and lipid-metabolism related regulation by the probiotics. Specific changes such as regulation of cell-cell adhesion by B. lactis 420, superoxide metabolism by L. salivarius Ls-33, and regulation of MAPK pathway by L. acidophilus NCFM™ were noted. Furthermore, fundamental differences were observed between the pathogenic and probiotic treatments in the Toll-like receptor pathway, especially for adapter molecules with a lowered level of transcriptional activation of MyD88, TRIF, IRAK1 and TRAF6 by probiotics compared to EHEC. The results in this study provide insights into the relationship between probiotics and human intestinal epithelial cells, notably with regard to strain-specific responses, and highlight the differences between transcriptional responses to pathogenic and probiotic bacteria.}, number={3}, journal={Beneficial Microbes}, publisher={Wageningen Academic Publishers}, author={Putaala, H. and Barrangou, R. and Leyer, G. and Ouwehand, A. and Hansen, E. Bech and Romero, D. and Rautonen, N.}, year={2010}, month={Sep}, pages={283–295} }
 @article{horvath_barrangou_2010, title={CRISPR/Cas, the Immune System of Bacteria and Archaea}, volume={327}, DOI={10.1126/science.1179555}, abstractNote={CRISPR Defenses
          
            Prokaryotes can be infected by parasites and pathogens and, like eukaryotes, have evolved systems to protect themselves.
            Horvath and Barrangou
            (p.
            167
            ) review a recently discovered prokaryotic “immune system” characterized by CRISPR—clustered regularly interspaced short palindromic repeats—found in most archaeal and many bacterial species. CRISPR loci harbor short sequences captured from viruses and invasive genetic elements. These sequences are transcribed, and the RNA is cleaved into short CRISPR RNAs (crRNAs) by one of a family of CRISPR-associated (cas) proteins. These crRNAs direct other cas family proteins to homologous nucleic acid targets to effect their destruction. Through its ability to impede the spread of specific nucleic acid sequences, the CRISPR/Cas systems might be exploited to block the dissemination of antibiotic-resistance markers.
          }, number={5962}, journal={Science}, publisher={American Association for the Advancement of Science (AAAS)}, author={Horvath, P. and Barrangou, R.}, year={2010}, month={Jan}, pages={167–170} }
 @article{duong_miller_barrangou_azcarate-peril_klaenhammer_2010, title={Construction of vectors for inducible and constitutive gene expression in Lactobacillus}, volume={4}, DOI={10.1111/j.1751-7915.2010.00200.x}, abstractNote={SummaryMicroarray analysis of the genome of Lactobacillus acidophilus identified a number of operons that were differentially expressed in response to carbohydrate source or constitutively expressed regardless of carbohydrate source. These included operons implicated in the transport and catabolism of fructooligosaccharides (FOS), lactose (lac), trehalose (tre) and genes directing glycolysis. Analysis of these operons identified a number of putative promoter and repressor elements, which were used to construct a series of expression vectors for use in lactobacilli, based on the broad host range pWV01 replicon. A β‐glucuronidase (GusA3) reporter gene was cloned into each vector to characterize expression from each promoter. GUS reporter assays showed FOS, lac and tre based vectors to be highly inducible by their specific carbohydrate and repressed by glucose. Additionally, a construct based on the phosphoglycerate mutase (pgm) promoter was constitutively highly expressed. To demonstrate the potential utility of these vectors, we constructed a plasmid for the overexpression of the oxalate degradation pathway (Frc and Oxc) of L. acidophilus NCFM. This construct was able to improve oxalate degradation by L. gasseri ATCC 33323 and compliment a L. acidophilus oxalate‐deficient mutant. Development of these expression vectors could support several novel applications, including the expression of enzymes, proteins, vaccines and biotherapeutics by intestinal lactobacilli.}, number={3}, journal={Microbial Biotechnology}, publisher={Wiley-Blackwell}, author={Duong, Tri and Miller, Michael J. and Barrangou, Rodolphe and Azcarate-Peril, M. Andrea and Klaenhammer, Todd R.}, year={2010}, month={Sep}, pages={357–367} }
 @article{garneau_dupuis_villion_romero_barrangou_boyaval_fremaux_horvath_magadán_moineau_2010, title={The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA}, volume={468}, DOI={10.1038/nature09523}, abstractNote={Bacteria and Archaea have developed several defence strategies against foreign nucleic acids such as viral genomes and plasmids. Among them, clustered regularly interspaced short palindromic repeats (CRISPR) loci together with cas (CRISPR-associated) genes form the CRISPR/Cas immune system, which involves partially palindromic repeats separated by short stretches of DNA called spacers, acquired from extrachromosomal elements. It was recently demonstrated that these variable loci can incorporate spacers from infecting bacteriophages and then provide immunity against subsequent bacteriophage infections in a sequence-specific manner. Here we show that the Streptococcus thermophilus CRISPR1/Cas system can also naturally acquire spacers from a self-replicating plasmid containing an antibiotic-resistance gene, leading to plasmid loss. Acquired spacers that match antibiotic-resistance genes provide a novel means to naturally select bacteria that cannot uptake and disseminate such genes. We also provide in vivo evidence that the CRISPR1/Cas system specifically cleaves plasmid and bacteriophage double-stranded DNA within the proto-spacer, at specific sites. Our data show that the CRISPR/Cas immune system is remarkably adapted to cleave invading DNA rapidly and has the potential for exploitation to generate safer microbial strains. CRISPR/Cas is a microbial immune system that is known to protect bacteria from viral infection. It is now shown that the Streptococcus thermophilus CRISPR/Cas system can prevent both plasmid carriage and phage infection through cleavage of invading double-stranded DNA of both viral and plasmid origin. The system seems remarkably adapted to this end, and it is thought that CRISPR/Cas could be used to naturally generate safer and more robust bacteria that are resistant to the acquisition and spread of antibiotic resistance genes. CRISPR/Cas is a microbial immune system that is known to protect bacteria from virus infection. These authors show that the Streptococcus thermophilus CRISPR/Cas system can prevent both plasmid carriage and phage infection through cleavage of invading double-stranded DNA.}, number={7320}, journal={Nature}, publisher={Springer Nature}, author={Garneau, Josiane E. and Dupuis, Marie-Ève and Villion, Manuela and Romero, Dennis A. and Barrangou, Rodolphe and Boyaval, Patrick and Fremaux, Christophe and Horvath, Philippe and Magadán, Alfonso H. and Moineau, Sylvain}, year={2010}, month={Nov}, pages={67–71} }
 @article{ventura_turroni_lima-mendez_foroni_zomer_duranti_giubellini_bottacini_horvath_barrangou_et al._2009, title={Comparative Analyses of Prophage-Like Elements Present in Bifidobacterial Genomes}, volume={75}, DOI={10.1128/aem.01112-09}, abstractNote={ABSTRACT
          
            So far, only scarce and rather diffuse information is available on bacteriophages infecting members of the genus
            Bifidobacterium
            . In the current study, we investigated the genetic organization, phylogenetic relationships, and, in some cases, transcription profiles and inducibility of 19 prophage-like elements present on the individual chromosomes of nine bifidobacterial strains, which represent six different species.
          }, number={21}, journal={Applied and Environmental Microbiology}, publisher={American Society for Microbiology}, author={Ventura, M. and Turroni, F. and Lima-Mendez, G. and Foroni, E. and Zomer, A. and Duranti, S. and Giubellini, V. and Bottacini, F. and Horvath, P. and Barrangou, R. and et al.}, year={2009}, month={Sep}, pages={6929–6936} }
 @article{barrangou_briczinski_traeger_loquasto_richards_horvath_coute-monvoisin_leyer_rendulic_steele_et al._2009, title={Comparison of the Complete Genome Sequences of Bifidobacterium animalis subsp. lactis DSM 10140 and Bl-04}, volume={191}, DOI={10.1128/jb.00155-09}, abstractNote={ABSTRACT
          
            Bifidobacteria are important members of the human gut flora, especially in infants. Comparative genomic analysis of two
            Bifidobacterium animalis
            subsp.
            lactis
            strains revealed evolution by internal deletion of consecutive spacer-repeat units within a novel clustered regularly interspaced short palindromic repeat locus, which represented the largest differential content between the two genomes. Additionally, 47 single nucleotide polymorphisms were identified, consisting primarily of nonsynonymous mutations, indicating positive selection and/or recent divergence. A particular nonsynonymous mutation in a putative glucose transporter was linked to a negative phenotypic effect on the ability of the variant to catabolize glucose, consistent with a modification in the predicted protein transmembrane topology. Comparative genome sequence analysis of three
            Bifidobacterium
            species provided a core genome set of 1,117 orthologs complemented by a pan-genome of 2,445 genes. The genome sequences of the intestinal bacterium
            B. animalis
            subsp.
            lactis
            provide insights into rapid genome evolution and the genetic basis for adaptation to the human gut environment, notably with regard to catabolism of dietary carbohydrates, resistance to bile and acid, and interaction with the intestinal epithelium. The high degree of genome conservation observed between the two strains in terms of size, organization, and sequence is indicative of a genomically monomorphic subspecies and explains the inability to differentiate the strains by standard techniques such as pulsed-field gel electrophoresis.
          }, number={13}, journal={Journal of Bacteriology}, publisher={American Society for Microbiology}, author={Barrangou, R. and Briczinski, E. P. and Traeger, L. L. and Loquasto, J. R. and Richards, M. and Horvath, P. and Coute-Monvoisin, A.-C. and Leyer, G. and Rendulic, S. and Steele, J. L. and et al.}, year={2009}, month={Apr}, pages={4144–4151} }
 @misc{barrangou_azcarate-peril_altermann_duong_klaenhammer_2009, title={Compositions comprising promoter sequences and methods of use}, volume={7,495,092}, number={2009 Feb. 24}, author={Barrangou, R. and Azcarate-Peril, A. and Altermann, E. and Duong, T. and Klaenhammer, T. R.}, year={2009} }
 @article{briczinski_loquasto_barrangou_dudley_roberts_roberts_2009, title={Strain-Specific Genotyping of Bifidobacterium animalis subsp. lactis by Using Single-Nucleotide Polymorphisms, Insertions, and Deletions}, volume={75}, DOI={10.1128/aem.01430-09}, abstractNote={ABSTRACT
          
            Several probiotic strains of
            Bifidobacterium animalis
            subsp.
            lactis
            are widely supplemented into food products and dietary supplements due to their documented health benefits and ability to survive within the mammalian gastrointestinal tract and acidified dairy products. The strain specificity of these characteristics demands techniques with high discriminatory power to differentiate among strains. However, to date, molecular approaches, such as pulsed-field gel electrophoresis and randomly amplified polymorphic DNA-PCR, have been ineffective at achieving strain separation due to the monomorphic nature of this subspecies. Previously, sequencing and comparison of two
            B. animalis
            subsp.
            lactis
            genomes (DSMZ 10140 and Bl-04) confirmed this high level of sequence similarity, identifying only 47 single-nucleotide polymorphisms (SNPs) and four insertions and/or deletions (INDELs) between them. In this study, we hypothesized that a sequence-based typing method targeting these loci would permit greater discrimination between strains than previously attempted methods. Sequencing 50 of these loci in 24 strains of
            B. animalis
            subsp.
            lactis
            revealed that a combination of nine SNPs/INDELs could be used to differentiate strains into 14 distinct genotypic groups. In addition, the presence of a nonsynonymous SNP within the gene encoding a putative glucose uptake protein was found to correlate with the ability of certain strains to transport glucose and to grow rapidly in a medium containing glucose as the sole carbon source. The method reported here can be used in clinical, regulatory, and commercial applications requiring identification of
            B. animalis
            subsp.
            lactis
            at the strain level.
          }, number={23}, journal={Applied and Environmental Microbiology}, publisher={American Society for Microbiology}, author={Briczinski, E. P. and Loquasto, J. R. and Barrangou, R. and Dudley, E. G. and Roberts, A. M. and Roberts, R. F.}, year={2009}, month={Oct}, pages={7501–7508} }
 @article{barrangou_horvath_2009, title={The CRISPR System Protects Microbes against Phages, Plasmids}, volume={4}, DOI={10.1128/microbe.4.224.1}, abstractNote={Many scientists believe that phages are the most abundant life form on Earth. Although phages outnumber their bacterial prey 10-fold, bacteria persist, sometimes relying on clustered regularly interspaced short palindromic repeats (CRISPRs) of DNA sequence as a defense mechanism. CRISPRs, first recognized in Escherichia coli in 1987, are found within the genomes of about 40% of bacteria and 90% of archaea tested so far.}, number={5}, journal={Microbe Magazine}, publisher={American Society for Microbiology}, author={Barrangou, Rodolphe and Horvath, Philippe}, year={2009}, month={May}, pages={224–230} }
 @article{azcarate-peril_altermann_goh_tallon_sanozky-dawes_pfeiler_o'flaherty_buck_dobson_duong_et al._2008, title={Analysis of the genome sequence of Lactobacillus gasseri ATCC 33323 reveals the molecular basis of an autochthonous intestinal organism}, volume={74}, ISSN={["1098-5336"]}, DOI={10.1128/aem.00054-08}, abstractNote={ABSTRACTThis study presents the complete genome sequence ofLactobacillus gasseriATCC 33323, a neotype strain of human origin and a native species found commonly in the gastrointestinal tracts of neonates and adults. The plasmid-free genome was 1,894,360 bp in size and predicted to encode 1,810 genes. The GC content was 35.3%, similar to the GC content of its closest relatives,L. johnsoniiNCC 533 (34%) andL. acidophilusNCFM (34%). Two identical copies of the prophage LgaI (40,086 bp), of the Sfi11-likeSiphoviridaephage family, were integrated tandomly in the chromosome. A number of unique features were identified in the genome ofL. gasserithat were likely acquired by horizontal gene transfer and may contribute to the survival of this bacterium in its ecological niche.L. gasseriencodes two restriction and modification systems, which may limit bacteriophage infection.L. gasserialso encodes an operon for production of heteropolysaccharides of high complexity. A unique alternative sigma factor was present similar to that ofB. caccaeATCC 43185, a bacterial species isolated from human feces. In addition,L. gasseriencoded the highest number of putative mucus-binding proteins (14) among lactobacilli sequenced to date. Selected phenotypic characteristics that were compared between ATCC 33323 and other humanL. gasseristrains included carbohydrate fermentation patterns, growth and survival in bile, oxalate degradation, and adhesion to intestinal epithelial cells, in vitro. The results from this study indicated high intraspecies variability from a genome encoding traits important for survival and retention in the gastrointestinal tract.}, number={15}, journal={APPLIED AND ENVIRONMENTAL MICROBIOLOGY}, publisher={American Society for Microbiology}, author={Azcarate-Peril, M. Andrea and Altermann, Eric and Goh, Yong Jun and Tallon, Richard and Sanozky-Dawes, Rosemary B. and Pfeiler, Erika A. and O'Flaherty, Sarah and Buck, B. Logan and Dobson, Alleson and Duong, Tri and et al.}, year={2008}, month={Aug}, pages={4610–4625} }
 @article{horvath_coûté-monvoisin_romero_boyaval_fremaux_barrangou_2009, title={Comparative analysis of CRISPR loci in lactic acid bacteria genomes}, volume={131}, DOI={10.1016/j.ijfoodmicro.2008.05.030}, abstractNote={Clustered regularly interspaced short palindromic repeats (CRISPR) are hypervariable loci widely distributed in bacteria and archaea, that provide acquired immunity against foreign genetic elements. Here, we investigate the occurrence of CRISPR loci in the genomes of lactic acid bacteria (LAB), including members of the Firmicutes and Actinobacteria phyla. A total of 102 complete and draft genomes across 11 genera were studied and 66 CRISPR loci were identified in 26 species. We provide a comparative analysis of the CRISPR/cas content and diversity across LAB genera and species for 37 sets of CRISPR loci. We analyzed CRISPR repeats, CRISPR spacers, leader sequences, and cas gene content, sequences and architecture. Interestingly, multiple CRISPR families were identified within Bifidobacterium, Lactobacillus and Streptococcus, and similar CRISPR loci were found in distant organisms. Overall, eight distinct CRISPR families were identified consistently across CRISPR repeats, cas gene content and architecture, and sequences of the universal cas1 gene. Since the clustering of the CRISPR families does not correlate with the classical phylogenetic tree, we hypothesize that CRISPR loci have been subjected to horizontal gene transfer and further evolved independently in select lineages, in part due to selective pressure resulting from phage predation. Globally, we provide additional insights into the origin and evolution of CRISPR loci and discuss their contribution to microbial adaptation.}, number={1}, journal={International Journal of Food Microbiology}, publisher={Elsevier BV}, author={Horvath, Philippe and Coûté-Monvoisin, Anne-Claire and Romero, Dennis A. and Boyaval, Patrick and Fremaux, Christophe and Barrangou, Rodolphe}, year={2009}, month={Apr}, pages={62–70} }
 @article{klaenhammer_altermann_pfeiler_buck_goh_o'flaherty_barrangou_duong_2008, title={Functional genomics of probiotic Lactobacilli}, volume={42}, ISSN={["0192-0790"]}, DOI={10.1097/MCG.0b013e31817da140}, abstractNote={Lactic acid bacteria (LAB) have been used in fermentation processes for millennia. Recent applications such as the use of living cultures as probiotics have significantly increased industrial interest. Related bacterial strains can differ significantly in their genotype and phenotype, and features from one bacterial strain or species cannot necessarily be applied to a related one. These strain or family-specific differences often represent unique and applicable traits. Since 2002, the complete genomes of 13 probiotic LABs have been published. The presentation will discuss these genomes and highlight probiotic traits that are predicted, or functionally linked to genetic content. We have conducted a comparative genomic analysis of 4 completely sequenced Lactobacillus strains versus 25 lactic acid bacterial genomes present in the public database at thetime of analysis. Using Differential Blast Analysis, each genome is compared with 3 other Lactobacillus and 25 other LAB genomes. Differential Blast Analysis highlighted strain-specific genes that were not represented in any other LAB used in this analysis and also identified group-specific genes shared within lactobacilli. Lactobacillus-specific genes include mucus-binding proteins involved in cell-adhesion and several transport systems for carbohydrates and amino acids. Comparative genomic analysis has identified gene targets in Lactobacillus acidophilus for functional analysis, including adhesion to mucin and intestinal epithelial cells, acid tolerance, bile tolerance, and quorum sensing. Whole genome transcriptional profiling of L. acidophilus, and isogenic mutants thereof, has revealed the impact of varying conditions (pH, bile, carbohydrates) and food matrices on the expression of genes important to probiotic-linked mechanisms.}, number={8}, journal={JOURNAL OF CLINICAL GASTROENTEROLOGY}, publisher={Ovid Technologies (Wolters Kluwer Health)}, author={Klaenhammer, Todd R. and Altermann, Eric and Pfeiler, Erika and Buck, Brock Logan and Goh, Yong-Jun and O'Flaherty, Sarah and Barrangou, Rodolphe and Duong, Tri}, year={2008}, month={Sep}, pages={S160–S162} }
 @misc{klaenhammer_altermann_barrangou_russell_duong_2008, title={Lactobacillus acidophilus nucleic acid sequences encoding carbohydrate utilization-related proteins and uses therefor}, volume={7,459,289}, number={2008 Dec. 2}, author={Klaenhammer, T. R. and Altermann, E. and Barrangou, R. and Russell, W. M. and Duong, T.}, year={2008} }
 @article{barrangou_fremaux_deveau_richards_boyaval_moineau_romero_horvath_2007, title={CRISPR Provides Acquired Resistance Against Viruses in Prokaryotes}, volume={315}, DOI={10.1126/science.1138140}, abstractNote={
            Clustered regularly interspaced short palindromic repeats (CRISPR) are a distinctive feature of the genomes of most Bacteria and Archaea and are thought to be involved in resistance to bacteriophages. We found that, after viral challenge, bacteria integrated new spacers derived from phage genomic sequences. Removal or addition of particular spacers modified the phage-resistance phenotype of the cell. Thus, CRISPR, together with associated
            cas
            genes, provided resistance against phages, and resistance specificity is determined by spacer-phage sequence similarity.
          }, number={5819}, journal={Science}, publisher={American Association for the Advancement of Science (AAAS)}, author={Barrangou, R. and Fremaux, C. and Deveau, H. and Richards, M. and Boyaval, P. and Moineau, S. and Romero, D. A. and Horvath, P.}, year={2007}, month={Mar}, pages={1709–1712} }
 @article{horvath_romero_coute-monvoisin_richards_deveau_moineau_boyaval_fremaux_barrangou_2007, title={Diversity, Activity, and Evolution of CRISPR Loci in Streptococcus thermophilus}, volume={190}, DOI={10.1128/jb.01415-07}, abstractNote={ABSTRACT
          
            Clustered regularly interspaced short palindromic repeats (CRISPR) are hypervariable loci widely distributed in prokaryotes that provide acquired immunity against foreign genetic elements. Here, we characterize a novel
            Streptococcus thermophilus
            locus, CRISPR3, and experimentally demonstrate its ability to integrate novel spacers in response to bacteriophage. Also, we analyze CRISPR diversity and activity across three distinct CRISPR loci in several
            S. thermophilus
            strains. We show that both CRISPR repeats and
            cas
            genes are locus specific and functionally coupled. A total of 124 strains were studied, and 109 unique spacer arrangements were observed across the three CRISPR loci. Overall, 3,626 spacers were analyzed, including 2,829 for CRISPR1 (782 unique), 173 for CRISPR2 (16 unique), and 624 for CRISPR3 (154 unique). Sequence analysis of the spacers revealed homology and identity to phage sequences (77%), plasmid sequences (16%), and
            S. thermophilus
            chromosomal sequences (7%). Polymorphisms were observed for the CRISPR repeats, CRISPR spacers,
            cas
            genes, CRISPR motif, locus architecture, and specific sequence content. Interestingly, CRISPR loci evolved both via polarized addition of novel spacers after exposure to foreign genetic elements and via internal deletion of spacers. We hypothesize that the level of diversity is correlated with relative CRISPR activity and propose that the activity is highest for CRISPR1, followed by CRISPR3, while CRISPR2 may be degenerate. Globally, the dynamic nature of CRISPR loci might prove valuable for typing and comparative analyses of strains and microbial populations. Also, CRISPRs provide critical insights into the relationships between prokaryotes and their environments, notably the coevolution of host and viral genomes.
          }, number={4}, journal={Journal of Bacteriology}, publisher={American Society for Microbiology}, author={Horvath, P. and Romero, D. A. and Coute-Monvoisin, A.-C. and Richards, M. and Deveau, H. and Moineau, S. and Boyaval, P. and Fremaux, C. and Barrangou, R.}, year={2007}, pages={1401–1412} }
 @article{klaenhammer_azcarate-peril_altermann_barrangou_2007, title={Influence of the dairy environment on gene expression and substrate(1-3)}, volume={137}, ISSN={["0022-3166"]}, DOI={10.1093/jn/137.3.748s}, abstractNote={Lactic acid bacteria (LAB) are widely used for the industrial production of fermented dairy products and form a group of related low-GC-content gram-positive bacteria. The major species used in dairy manufacturing are Lactobacillus, Lactococcus, Streptococcus, and Leuconostoc. Traditionally most are applied as starter cultures for dairy fermentations or used as probiotic cultures, delivered in dairy vehicles. The appearance of the genomes of Lactococcus lactis, Bidifobacterium longum, Lactobacillus plantarum, L. johnsonii, L. acidophilus, 2 strains of Streptococcus thermophilus, and pending completion of many draft genomic sequences, is now promoting in-depth investigation into the comparative genetic content of LAB. Moreover, whole-genome transcriptional arrays are quickly revealing critical genes/operons that are coordinately expressed and the impact of environmental factors on expression of multiple gene sets. Comparative genomics between multiple genomes is providing insights into genes that are important in metabolic, physiological, and functional roles for different LAB in the environments they inhabit, ranging from the gastrointestinal tract to milk and acidified dairy products.}, number={3}, journal={JOURNAL OF NUTRITION}, author={Klaenhammer, Todd R. and Azcarate-Peril, M. Andrea and Altermann, Eric and Barrangou, Rodolphe}, year={2007}, month={Mar}, pages={748S–750S} }
 @article{deveau_barrangou_garneau_labonte_fremaux_boyaval_romero_horvath_moineau_2007, title={Phage Response to CRISPR-Encoded Resistance in Streptococcus thermophilus}, volume={190}, DOI={10.1128/jb.01412-07}, abstractNote={ABSTRACT
          
            Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated genes are linked to a mechanism of acquired resistance against bacteriophages. Bacteria can integrate short stretches of phage-derived sequences (spacers) within CRISPR loci to become phage resistant. In this study, we further characterized the efficiency of CRISPR1 as a phage resistance mechanism in
            Streptococcus thermophilus
            . First, we show that CRISPR1 is distinct from previously known phage defense systems and is effective against the two main groups of
            S. thermophilus
            phages. Analyses of 30 bacteriophage-insensitive mutants of
            S. thermophilus
            indicate that the addition of one new spacer in CRISPR1 is the most frequent outcome of a phage challenge and that the iterative addition of spacers increases the overall phage resistance of the host. The added new spacers have a size of between 29 to 31 nucleotides, with 30 being by far the most frequent. Comparative analysis of 39 newly acquired spacers with the complete genomic sequences of the wild-type phages 2972, 858, and DT1 demonstrated that the newly added spacer must be identical to a region (named proto-spacer) in the phage genome to confer a phage resistance phenotype. Moreover, we found a CRISPR1-specific sequence (NNAGAAW) located downstream of the proto-spacer region that is important for the phage resistance phenotype. Finally, we show through the analyses of 20 mutant phages that virulent phages are rapidly evolving through single nucleotide mutations as well as deletions, in response to CRISPR1.
          }, number={4}, journal={Journal of Bacteriology}, publisher={American Society for Microbiology}, author={Deveau, H. and Barrangou, R. and Garneau, J. E. and Labonte, J. and Fremaux, C. and Boyaval, P. and Romero, D. A. and Horvath, P. and Moineau, S.}, year={2007}, pages={1390–1400} }
 @article{duong_barrangou_russell_klaenhammer_2006, title={Characterization of the tre locus and analysis of trehalose cryoprotection in Lactobacillus acidophilus NCFM}, volume={72}, ISSN={["1098-5336"]}, DOI={10.1128/AEM.72.2.1218-1225.2006}, abstractNote={ABSTRACT
          
            Freezing and lyophilization are common methods used for preservation and storage of microorganisms during the production of concentrated starter cultures destined for industrial fermentations or product formulations. The compatible solute trehalose has been widely reported to protect bacterial, yeast and animal cells against a variety of environmental stresses, particularly freezing and dehydration. Analysis of the
            Lactobacillus acidophilus
            NCFM genome revealed a putative trehalose utilization locus consisting of a transcriptional regulator,
            treR
            ; a trehalose phosphoenolpyruvate transferase system (PTS) transporter,
            treB
            ; and a trehalose-6-phosphate hydrolase,
            treC
            . The objective of this study was to characterize the
            tre
            locus in
            L. acidophilus
            and determine whether or not intracellular uptake of trehalose contributes to cryoprotection. Cells subjected to repeated freezing and thawing cycles were monitored for survival in the presence of various concentrations of trehalose. At 20% trehalose a 2-log increase in survival was observed. The trehalose PTS transporter and trehalose hydrolase were disrupted by targeted plasmid insertions. The resulting mutants were unable to grow on trehalose, indicating that both trehalose transport into the cell via a PTS and hydrolysis via a trehalose-6-phosphate hydrolase were necessary for trehalose fermentation. Trehalose uptake was found to be significantly reduced in the transporter mutant but unaffected in the hydrolase mutant. Additionally, the cryoprotective effect of trehalose was reduced in these mutants, suggesting that intracellular transport and hydrolysis contribute significantly to cryoprotection.
          }, number={2}, journal={APPLIED AND ENVIRONMENTAL MICROBIOLOGY}, publisher={American Society for Microbiology}, author={Duong, T and Barrangou, R and Russell, WM and Klaenhammer, TR}, year={2006}, month={Feb}, pages={1218–1225} }
 @article{ventura_canchaya_bernini_altermann_barrangou_mcgrath_claesson_li_leahy_walker_et al._2006, title={Comparative genomics and transcriptional analysis of prophages identified in the Genomes of Lactobacillus gasseri, Lactobacillus salivarius, and Lactobacillus casei}, volume={72}, ISSN={["1098-5336"]}, DOI={10.1128/AEM.72.5.3130-3146.2006}, abstractNote={ABSTRACT
          
            Lactobacillus
gasseri
            ATCC 33323,
            Lactobacillus salivarius
            subsp.
            salivarius
            UCC 118, and
            Lactobacillus casei
            ATCC 334
contain one (LgaI), four (Sal1, Sal2, Sal3, Sal4), and one (Lca1)
distinguishable prophage sequences, respectively. Sequence analysis
revealed that LgaI, Lca1, Sal1, and Sal2 prophages belong to the group
of Sfi11-like
            pac
            site and
            cos
            site
            Siphoviridae
            , respectively. Phylogenetic investigation of
these newly described prophage sequences revealed that they have not
followed an evolutionary development similar to that of their bacterial
hosts and that they show a high degree of diversity, even within a
species. The attachment sites were determined for all these prophage
elements; LgaI as well as Sal1 integrates in tRNA genes, while prophage
Sal2 integrates in a predicted arginino-succinate lyase-encoding gene.
In contrast, Lca1 and the Sal3 and Sal4 prophage remnants are
integrated in noncoding regions in the
            L. casei
            ATCC 334 and
            L. salivarius
            UCC 118 genomes. Northern analysis showed that
large parts of the prophage genomes are transcriptionally silent and
that transcription is limited to genome segments located near the
attachment site. Finally, pulsed-field gel electrophoresis followed by
Southern blot hybridization with specific prophage probes indicates
that these prophage sequences are narrowly distributed within
lactobacilli.
          }, number={5}, journal={APPLIED AND ENVIRONMENTAL MICROBIOLOGY}, publisher={American Society for Microbiology}, author={Ventura, Marco and Canchaya, Carlos and Bernini, Valentina and Altermann, Eric and Barrangou, Rodolphe and McGrath, Stephen and Claesson, Marcus J. and Li, Yin and Leahy, Sinead and Walker, Carey D. and et al.}, year={2006}, month={May}, pages={3130–3146} }
 @article{makarova_slesarev_wolf_sorokin_mirkin_koonin_pavlov_pavlova_karamychev_polouchine_et al._2006, title={Comparative genomics of the lactic acid bacteria}, volume={103}, ISSN={["0027-8424"]}, DOI={10.1073/pnas.0607117103}, abstractNote={Lactic acid-producing bacteria are associated with various plant and animal niches and play a key role in the production of fermented foods and beverages. We report nine genome sequences representing the phylogenetic and functional diversity of these bacteria. The small genomes of lactic acid bacteria encode a broad repertoire of transporters for efficient carbon and nitrogen acquisition from the nutritionally rich environments they inhabit and reflect a limited range of biosynthetic capabilities that indicate both prototrophic and auxotrophic strains. Phylogenetic analyses, comparison of gene content across the group, and reconstruction of ancestral gene sets indicate a combination of extensive gene loss and key gene acquisitions via horizontal gene transfer during the coevolution of lactic acid bacteria with their habitats.}, number={42}, journal={PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA}, publisher={Proceedings of the National Academy of Sciences}, author={Makarova, K. and Slesarev, A. and Wolf, Y. and Sorokin, A. and Mirkin, B. and Koonin, E. and Pavlov, A. and Pavlova, N. and Karamychev, V. and Polouchine, N. and et al.}, year={2006}, month={Oct}, pages={15611–15616} }
 @article{barrangou_azcarate-peril_duong_conners_kelly_klaenhammer_2006, title={Global analysis of carbohydrate utilization by Lactobacillus acidophilus using cDNA microarrays}, volume={103}, ISSN={["0027-8424"]}, DOI={10.1073/pnas.0511287103}, abstractNote={
            The transport and catabolic machinery involved in carbohydrate utilization by
            Lactobacillus acidophilus
            was characterized genetically by using whole-genome cDNA microarrays. Global transcriptional profiles were determined for growth on glucose, fructose, sucrose, lactose, galactose, trehalose, raffinose, and fructooligosaccharides. Hybridizations were carried out by using a round-robin design, and microarray data were analyzed with a two-stage mixed model ANOVA. Differentially expressed genes were visualized by hierarchical clustering, volcano plots, and contour plots. Overall, only 63 genes (3% of the genome) showed a >4-fold induction. Specifically, transporters of the phospho
            enol
            pyruvate:sugar transferase system were identified for uptake of glucose, fructose, sucrose, and trehalose, whereas ATP-binding cassette transporters were identified for uptake of raffinose and fructooligosaccharides. A member of the LacS subfamily of galactoside-pentose hexuronide translocators was identified for uptake of galactose and lactose. Saccharolytic enzymes likely involved in the metabolism of monosaccharides, disaccharides, and polysaccharides into substrates of glycolysis were also found, including enzymatic machinery of the Leloir pathway. The transcriptome appeared to be regulated by carbon catabolite repression. Although substrate-specific carbohydrate transporters and hydrolases were regulated at the transcriptional level, genes encoding regulatory proteins CcpA, Hpr, HprK/P, and EI were consistently highly expressed. Genes central to glycolysis were among the most highly expressed in the genome. Collectively, microarray data revealed that coordinated and regulated transcription of genes involved in sugar uptake and metabolism is based on the specific carbohydrate provided.
            L. acidophilus
            's adaptability to environmental conditions likely contributes to its competitive ability for limited carbohydrate sources available in the human gastrointestinal tract.
          }, number={10}, journal={PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA}, publisher={Proceedings of the National Academy of Sciences}, author={Barrangou, R and Azcarate-Peril, MA and Duong, T and Conners, SB and Kelly, RM and Klaenhammer, TR}, year={2006}, month={Mar}, pages={3816–3821} }
 @article{altermann_russell_azcarate-peril_barrangou_buck_mcauliffe_souther_dobson_duong_callanan_et al._2005, title={Complete genome sequence of the probiotic lactic acid bacterium Lactobacillus acidophilus NCFM}, volume={102}, ISSN={["0027-8424"]}, DOI={10.1073/pnas.0409188102}, abstractNote={Lactobacillus acidophilusNCFM is a probiotic bacterium that has been produced commercially since 1972. The complete genome is 1,993,564 nt and devoid of plasmids. The average GC content is 34.71% with 1,864 predicted ORFs, of which 72.5% were functionally classified. Nine phage-related integrases were predicted, but no complete prophages were found. However, three unique regions designated as potential autonomous units (PAUs) were identified. These units resemble a unique structure and bear characteristics of both plasmids and phages. Analysis of the three PAUs revealed the presence of two R/M systems and a prophage maintenance system killer protein. A spacers interspersed direct repeat locus containing 32 nearly perfect 29-bp repeats was discovered and may provide a unique molecular signature for this organism.In silicoanalyses predicted 17 transposase genes and a chromosomal locus for lactacin B, a class II bacteriocin. Several mucus- and fibronectin-binding proteins, implicated in adhesion to human intestinal cells, were also identified. Gene clusters for transport of a diverse group of carbohydrates, including fructooligosaccharides and raffinose, were present and often accompanied by transcriptional regulators of the lacI family. For protein degradation and peptide utilization, the organism encoded 20 putative peptidases, homologs for PrtP and PrtM, and two complete oligopeptide transport systems. Nine two-component regulatory systems were predicted, some associated with determinants implicated in bacteriocin production and acid tolerance. Collectively, these features within the genome sequence ofL. acidophilusare likely to contribute to the organisms' gastric survival and promote interactions with the intestinal mucosa and microbiota.}, number={11}, journal={PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA}, publisher={Proceedings of the National Academy of Sciences}, author={Altermann, E and Russell, WM and Azcarate-Peril, MA and Barrangou, R and Buck, BL and McAuliffe, O and Souther, N and Dobson, A and Duong, T and Callanan, M and et al.}, year={2005}, month={Mar}, pages={3906–3912} }
 @article{klaenhammer_peril_barrangou_duong_altermann_2005, title={Genomic Perspectives on Probiotic Lactic Acid Bacteria}, volume={24}, ISBN={1342-1441}, DOI={10.12938/bifidus.24.31}, abstractNote={The lactic acid bacteria are Gram-positive fermentative microorganisms known primarily for their roles as starter cultures and probiotics. The food industry represents one of the largest manufacturing industries in the world and recent trends are rapidly expanding the use of probiotic cultures within functional foods. Understanding and control of lactic acid bacteria is now being revolutionized by genomic sciences and the appearance of the complete genome sequences for Bifidobacterium longum, Lactobacillus johnsonii, Lactobacillus plantarum, and draft sequences for Lactobacillus gasseri and Lactobacillus casei. This explosion of DNA sequence information, accompanied by the development of bioinformatic tools for nucleic acid and protein analysis, now allows rapid characterization of the lactic acid bacteria for their genomic content and expression profiles across the entire genome. Comparative genomics has already revealed important similarities and differences in strains, species, and genera and will likely identify key genetic features responsible for the beneficial properties ascribed to probiotic lactic acid bacteria. Practical genomics for the lactic acid bacteria promises to establish the genetic landscape, correlate genotypes with desirable phenotypes, establish genetic criteria for strain selection, improve culture stability by stress preconditioning, provide opportunities for metabolic engineering, and uncover a mechanistic basis for the beneficial activities of probiotics when delivered in various foods. This presentation will examine the genomic content of probiotic Lactobacillus cultures, compared to those lactic acid bacterial genomes that have appeared recently. In addition, expression profiling by whole genome microarrays will be used to illustrate how environmental conditions encountered during biomanufacturing, fermentation, and the gastrointestinal tract can impact gene expression and culture functionality.}, number={2}, journal={Bioscience and Microflora}, publisher={BMFH Press}, author={Klaenhammer, Todd R. and Peril, Andrea Azcarate and Barrangou, Rodolphe and Duong, Tri and Altermann, Eric}, year={2005}, pages={31–33} }
 @misc{klaenhammer_barrangou_buck_azcarate-peril_altermann_2005, title={Genomic features of lactic acid bacteria effecting bioprocessing and health}, volume={29}, ISSN={["1574-6976"]}, DOI={10.1016/j.femsre.2005.04.007}, abstractNote={The lactic acid bacteria are a functionally related group of organisms known primarily for their bioprocessing roles in food and beverages. More recently, selected members of the lactic acid bacteria have been implicated in a number of probiotic roles that impact general health and well-being. Genomic analyses of multiple members of the lactic acid bacteria, at the genus, species, and strain level, have now elucidated many genetic features that direct their fermentative and probiotic roles. This information is providing an important platform for understanding core mechanisms that control and regulate bacterial growth, survival, signaling, and fermentative processes and, in some cases, potentially underlying probiotic activities within complex microbial and host ecosystems.}, number={3}, journal={FEMS MICROBIOLOGY REVIEWS}, publisher={Wiley-Blackwell}, author={Klaenhammer, TR and Barrangou, R and Buck, BL and Azcarate-Peril, MA and Altermann, E}, year={2005}, month={Aug}, pages={393–409} }
 @article{klaenhammer_barrangou_buck_azcarate-peril_altermann_2005, title={Genomic features of lactic acid bacteria effecting bioprocessing and health}, volume={29}, DOI={10.1016/j.fmrre.2005.04.007}, abstractNote={The lactic acid bacteria are a functionally related group of organisms known primarily for their bioprocessing roles in food and beverages. More recently, selected members of the lactic acid bacteria have been implicated in a number of probiotic roles that impact general health and well-being. Genomic analyses of multiple members of the lactic acid bacteria, at the genus, species, and strain level, have now elucidated many genetic features that direct their fermentative and probiotic roles. This information is providing an important platform for understanding core mechanisms that control and regulate bacterial growth, survival, signaling, and fermentative processes and, in some cases, potentially underlying probiotic activities within complex microbial and host ecosystems.}, number={3}, journal={FEMS Microbiology Reviews}, publisher={Oxford University Press (OUP)}, author={Klaenhammer, Todd R. and Barrangou, Rodolphe and Buck, B. Logan and Azcarate-Peril, M. Andrea and Altermann, Eric}, year={2005}, month={Aug}, pages={393–409} }
 @misc{barrangou_klaenhammer_altermann_2004, title={Lactobacillus acidophilus nucleic acids encoding fructo-oligosaccharide utilization compounds and uses thereof}, volume={7,407,787}, number={2004 Jun 22}, publisher={Washington, DC: U.S. Patent and Trademark Office}, author={Barrangou, R. and Klaenhammer, T. R. and Altermann, E.}, year={2004} }
 @article{pridmore_berger_desiere_vilanova_barretto_pittet_zwahlen_rouvet_altermann_barrangou_et al._2004, title={The genome sequence of the probiotic intestinal bacterium Lactobacillus johnsonii NCC 533}, volume={101}, ISSN={["0027-8424"]}, DOI={10.1073/pnas.0307327101}, abstractNote={
            Lactobacillus johnsonii
            NCC 533 is a member of the acidophilus group of intestinal lactobacilli that has been extensively studied for their “probiotic” activities that include, pathogen inhibition, epithelial cell attachment, and immunomodulation. To gain insight into its physiology and identify genes potentially involved in interactions with the host, we sequenced and analyzed the 1.99-Mb genome of
            L. johnsonii
            NCC 533. Strikingly, the organism completely lacked genes encoding biosynthetic pathways for amino acids, purine nucleotides, and most cofactors. In apparent compensation, a remarkable number of uncommon and often duplicated amino acid permeases, peptidases, and phosphotransferase-type transporters were discovered, suggesting a strong dependency of NCC 533 on the host or other intestinal microbes to provide simple monomeric nutrients. Genome analysis also predicted an abundance (>12) of large and unusual cell-surface proteins, including fimbrial subunits, which may be involved in adhesion to glycoproteins or other components of mucin, a characteristic expected to affect persistence in the gastrointestinal tract (GIT). Three bile salt hydrolases and two bile acid transporters, proteins apparently critical for GIT survival, were also detected.
            In silico
            genome comparisons with the >95% complete genome sequence of the closely related
            Lactobacillus gasseri
            revealed extensive synteny punctuated by clear-cut insertions or deletions of single genes or operons. Many of these regions of difference appear to encode metabolic or structural components that could affect the organisms competitiveness or interactions with the GIT ecosystem.
          }, number={8}, journal={PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA}, publisher={Proceedings of the National Academy of Sciences}, author={Pridmore, RD and Berger, B and Desiere, F and Vilanova, D and Barretto, C and Pittet, AC and Zwahlen, MC and Rouvet, M and Altermann, E and Barrangou, R and et al.}, year={2004}, month={Feb}, pages={2512–2517} }
 @article{barrangou_altermann_hutkins_cano_klaenhammer_2003, title={Functional and comparative genomic analyses of an operon involved in fructooligosaccharide utilization by Lactobacillus acidophilus}, volume={100}, ISSN={["0027-8424"]}, DOI={10.1073/pnas.1332765100}, abstractNote={
            Lactobacillus acidophilus
            is a probiotic organism that displays
 the ability to use prebiotic compounds such as fructooligosaccharides (FOS),
 which stimulate the growth of beneficial commensals in the gastrointestinal
 tract. However, little is known about the mechanisms and genes involved in FOS
 utilization by
            Lactobacillus
            species. Analysis of the
            L.
 acidophilus
            NCFM genome revealed an
            msm
            locus composed of a
 transcriptional regulator of the
            Lac
            I family, a four-component
 ATP-binding cassette (ABC) transport system, a fructosidase, and a sucrose
 phosphorylase. Transcriptional analysis of this operon demonstrated that gene
 expression was induced by sucrose and FOS but not by glucose or fructose,
 suggesting some specificity for nonreadily fermentable sugars. Additionally,
 expression was repressed by glucose but not by fructose, suggesting catabolite
 repression via two
            cre
            -like sequences identified in the
 promoter–operator region. Insertional inactivation of the genes encoding
 the ABC transporter substrate-binding protein and the fructosidase reduced the
 ability of the mutants to grow on FOS. Comparative analysis of gene
 architecture within this cluster revealed a high degree of synteny with
 operons in
            Streptococcus mutans
            and
            Streptococcus
 pneumoniae
            . However, the association between a fructosidase and an ABC
 transporter is unusual and may be specific to
            L. acidophilus
            . This is
 a description of a previously undescribed gene locus involved in transport and
 catabolism of FOS compounds, which can promote competition of beneficial
 microorganisms in the human gastrointestinal tract.
          }, number={15}, journal={PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA}, publisher={Proceedings of the National Academy of Sciences}, author={Barrangou, R and Altermann, E and Hutkins, R and Cano, R and Klaenhammer, TR}, year={2003}, month={Jul}, pages={8957–8962} }
 @article{barrangou_yoon_breidt_fleming_klaenhammer_2002, title={Characterization of six Leuconostoc fallax bacteriophages isolated from an industrial sauerkraut fermentation}, volume={68}, ISSN={["1098-5336"]}, DOI={10.1128/aem.68.11.5452-5458.2002}, abstractNote={ABSTRACTSix bacteriophages active againstLeuconostoc fallaxstrains were isolated from industrial sauerkraut fermentation brines. These phages were characterized as to host range, morphology, structural proteins, and genome fingerprint. They were exclusively lytic against the speciesL. fallaxand had different host ranges among the strains of this species tested. Morphologically, three of the phages were assigned to the familySiphoviridae, and the three others were assigned to the familyMyoviridae. Major capsid proteins detected by electrophoresis were distinct for each of the two morphotypes. Restriction fragment length polymorphism analysis and randomly amplified polymorphic DNA fingerprinting showed that all six phages were genetically distinct. These results revealed for the first time the existence of bacteriophages that are active againstL. fallaxand confirmed the presence and diversity of bacteriophages in a sauerkraut fermentation. Since a variety ofL. fallaxstrains have been shown to be present in sauerkraut fermentation, bacteriophages active againstL. fallaxare likely to contribute to the microbial ecology of sauerkraut fermentation and could be responsible for some of the variability observed in this type of fermentation.}, number={11}, journal={APPLIED AND ENVIRONMENTAL MICROBIOLOGY}, publisher={American Society for Microbiology}, author={Barrangou, R and Yoon, SS and Breidt, F and Fleming, HP and Klaenhammer, TR}, year={2002}, month={Nov}, pages={5452–5458} }
 @article{barrangou_yoon_breidt_fleming_klaenhammer_2002, title={Identification and characterization of Leuconostoc fallax strains isolated from an industrial sauerkraut fermentation}, volume={68}, ISSN={["0099-2240"]}, DOI={10.1128/aem.68.6.2877-2884.2002}, abstractNote={ABSTRACT
          
            Lactic acid bacterial strains were isolated from brines sampled after 7 days of an industrial sauerkraut fermentation, and six strains were selected on the basis of susceptibility to bacteriophages. Bacterial growth in cabbage juice was monitored, and the fermentation end products were identified, quantified, and compared to those of
            Leuconostoc mesenteroides
            . Identification by biochemical fingerprinting, endonuclease digestion of the 16S-23S intergenic transcribed spacer region, and sequencing of variable regions V1 and V2 of the 16S rRNA gene indicated that the six selected sauerkraut isolates were
            Leuconostoc fallax
            strains. Random amplification of polymorphic DNA fingerprints indicated that the strains were distinct from one another. The growth and fermentation patterns of the
            L. fallax
            isolates were highly similar to those of
            L. mesenteroides
            . The final pH of cabbage juice fermentation was 3.6, and the main fermentation end products were lactic acid, acetic acid, and mannitol for both species. However, none of the
            L. fallax
            strains exhibited the malolactic reaction, which is characteristic of most
            L. mesenteroides
            strains. These results indicated that in addition to
            L. mesenteroides
            , a variety of
            L. fallax
            strains may be present in the heterofermentative stage of sauerkraut fermentation. The microbial ecology of sauerkraut fermentation appears to be more complex than previously indicated, and the prevalence and roles of
            L. fallax
            require further investigation.
          }, number={6}, journal={APPLIED AND ENVIRONMENTAL MICROBIOLOGY}, publisher={American Society for Microbiology}, author={Barrangou, R and Yoon, SS and Breidt, F and Fleming, HP and Klaenhammer, TR}, year={2002}, month={Jun}, pages={2877–2884} }
 @article{yoon_barrangou-poueys_breidt_klaenhammer_fleming_2002, title={Isolation and characterization of bacteriophages from fermenting sauerkraut}, volume={68}, ISSN={["0099-2240"]}, DOI={10.1128/aem.68.2.973-976.2002}, abstractNote={ABSTRACT
          
            This paper presents the first report of bacteriophage isolated from commercial vegetable fermentations. Nine phages were isolated from two 90-ton commercial sauerkraut fermentations. These phages were active against fermentation isolates and selected
            Leuconostoc mesenteroides
            and
            Lactobacillus plantarum
            strains, including a starter culture. Phages were characterized as members of the
            Siphoviridae
            and
            Myoviridae
            families. All
            Leuconostoc
            phages reported previously, primarily of dairy origin, belonged to the
            Siphoviridae
            family.
          }, number={2}, journal={APPLIED AND ENVIRONMENTAL MICROBIOLOGY}, publisher={American Society for Microbiology}, author={Yoon, SS and Barrangou-Poueys, R and Breidt, F and Klaenhammer, TR and Fleming, HP}, year={2002}, month={Feb}, pages={973–976} }