@article{collias_leenay_slotkowski_zuo_collins_mcgirr_liu_beisel_2020, title={A positive, growth-based PAM screen identifies noncanonical motifs recognized by the S. pyogenes Cas9}, volume={6}, ISBN={2375-2548}, DOI={10.1126/sciadv.abb4054}, abstractNote={SpyCas9 and its engineered variants can recognize NYGG PAMs, affecting their use for genome editing and off-target predictions.}, number={29}, journal={SCIENCE ADVANCES}, author={Collias, D. and Leenay, R. T. and Slotkowski, R. A. and Zuo, Z. and Collins, S. P. and McGirr, B. A. and Liu, J. and Beisel, C. L.}, year={2020}, month={Jul} }
 @article{leenay_vento_shah_martino_leulier_beisel_2019, title={Genome Editing with CRISPR-Cas9 in Lactobacillus plantarum Revealed That Editing Outcomes Can Vary Across Strains and Between Methods}, volume={14}, ISSN={["1860-7314"]}, DOI={10.1002/biot.201700583}, abstractNote={Lactic-acid bacteria such as Lactobacillus plantarum are commonly used for fermenting foods and as probiotics, where increasingly sophisticated genome-editing tools are employed to elucidate and enhance these microbes' beneficial properties. The most advanced tools to date utilize an oligonucleotide or double-stranded DNA donor for recombineering and Cas9 for targeted DNA cleavage. As the associated methods are often developed in isolation for one strain, it remains unclear how different Cas9-based editing methods compare across strains. Here, this work directly compares two methods in different strains of L. plantarum: one utilizing a plasmid-encoded recombineering template and another utilizing an oligonucleotide donor and an inducible DNA recombinase. This comparison reveals one instance in which only the recombineering-template method generates desired edits and another instance in which only the oligo method generates desired edits. It is further found that both methods exhibit highly variable success editing the same site across multiple L. plantarum strains. Finally, failure modes are identified for the recombineering-template method, including a consistent genomic deletion and reversion of a point mutation in the recombineering template. This study therefore highlights surprising differences for Cas9-mediated genome editing between methods and related strains, arguing for the need for multiple, distinct methods when performing CRISPR-based editing in bacteria.}, number={3}, journal={BIOTECHNOLOGY JOURNAL}, author={Leenay, Ryan T. and Vento, Justin M. and Shah, Malay and Martino, Maria Elena and Leulier, Francois and Beisel, Chase L.}, year={2019}, month={Mar} }
 @article{liao_ttofali_slotkowski_denny_cecil_leenay_keung_beisel_2019, title={Modular one-pot assembly of CRISPR arrays enables library generation and reveals factors influencing crRNA biogenesis}, volume={10}, ISSN={["2041-1723"]}, DOI={10.1038/s41467-019-10747-3}, abstractNote={Abstract}, journal={NATURE COMMUNICATIONS}, author={Liao, Chunyu and Ttofali, Fani and Slotkowski, Rebecca A. and Denny, Steven R. and Cecil, Taylor D. and Leenay, Ryan T. and Keung, Albert J. and Beisel, Chase L.}, year={2019}, month={Jul} }
 @misc{leenay_beisel_2017, title={Deciphering, Communicating, and Engineering the CRISPR PAM}, volume={429}, ISSN={["1089-8638"]}, DOI={10.1016/j.jmb.2016.11.024}, abstractNote={Clustered regularly interspaced short palindromic repeat (CRISPR) loci and their flanking CRISPR-associated (cas) genes make up RNA-guided, adaptive immune systems in prokaryotes whose effector proteins have become powerful tools for basic research and biotechnology. While the Cas effector proteins are remarkably diverse, they commonly rely on protospacer-adjacent motifs (PAMs) as the first step in target recognition. PAM sequences are known to vary considerably between systems and have proven to be difficult to predict, spurring the need for new tools to rapidly identify and communicate these sequences. Recent advances have also shown that Cas proteins can be engineered to alter PAM recognition, opening new opportunities to develop CRISPR-based tools with enhanced targeting capabilities. In this review, we discuss the properties of the CRISPR PAM and the emerging tools for determining, visualizing, and engineering PAM recognition. We also propose a standard means of orienting the PAM to simplify how its location and sequence are communicated.}, number={2}, journal={JOURNAL OF MOLECULAR BIOLOGY}, author={Leenay, Ryan T. and Beisel, Chase L.}, year={2017}, month={Jan}, pages={177–191} }
 @misc{luo_leenay_beisel_2016, title={Current and future prospects for CRISPR-based tools in bacteria}, volume={113}, ISSN={["1097-0290"]}, DOI={10.1002/bit.25851}, abstractNote={ABSTRACT}, number={5}, journal={BIOTECHNOLOGY AND BIOENGINEERING}, author={Luo, Michelle L. and Leenay, Ryan T. and Beisel, Chase L.}, year={2016}, month={May}, pages={930–943} }
 @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{luo_mullis_leenay_beisel_2015, title={Repurposing endogenous type I CRISPR-Cas systems for programmable gene repression}, volume={43}, ISSN={["1362-4962"]}, DOI={10.1093/nar/gku971}, abstractNote={CRISPR-Cas systems have shown tremendous promise as heterologous tools for genome editing and transcriptional regulation. Because these RNA-directed immune systems are found in most prokaryotes, an opportunity exists to harness the endogenous systems as convenient tools in these organisms. Here, we report that the Type I-E CRISPR-Cas system in Escherichia coli can be co-opted for programmable transcriptional repression. We found that deletion of the signature cas3 gene converted this immune system into a programmable gene regulator capable of reversible gene silencing of heterologous and endogenous genes. Targeting promoter regions yielded the strongest repression, whereas targeting coding regions showed consistent strand bias. Furthermore, multi-targeting CRISPR arrays could generate complex phenotypes. This strategy offers a simple approach to convert many endogenous Type I systems into transcriptional regulators, thereby expanding the available toolkit for CRISPR-mediated genetic control while creating new opportunities for genome-wide screens and pathway engineering.}, number={1}, journal={NUCLEIC ACIDS RESEARCH}, author={Luo, Michelle L. and Mullis, Adam S. and Leenay, Ryan T. and Beisel, Chase L.}, year={2015}, month={Jan}, pages={674–681} }