@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_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} }
 @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{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{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{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} }
 @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{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={<h2>Summary</h2> 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 <i>Bacillus halodurans</i> I-C (Cascade), <i>Escherichia coli</i> I-E (Cascade), <i>Streptococcus thermophilus</i> II-A CRISPR1 (Cas9), and <i>Francisella novicida</i> 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{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={Abstract}, 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} }
 @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{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={The 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.}, 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{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}, 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} }