@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}, journal={NUCLEIC ACIDS RESEARCH}, author={Roberts, Avery and Nethery, Matthew A. and Barrangou, Rodolphe}, year={2022}, month={Nov} }
 @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{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, blaCMY-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 blaCMY-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{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{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
              }, 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} }
 @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} }