title: Identifying and Visualizing Functional PAM Diversity across CRISPR-Cas Systems.
abstract: 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.
authors.affiliation: Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, NC 27695, USA.
authors.forename: Ryan T
authors.initials: RT
authors.lastname: Leenay
authors.affiliation: Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, NC 27695, USA.
authors.forename: Kenneth R
authors.initials: KR
authors.lastname: Maksimchuk
authors.affiliation: Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, NC 27695, USA.
authors.forename: Rebecca A
authors.initials: RA
authors.lastname: Slotkowski
authors.affiliation: Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, NC 27695, USA.
authors.forename: Roma N
authors.initials: RN
authors.lastname: Agrawal
authors.affiliation: Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, NC 27695, USA; Chemical Engineering Department, Faculty of Engineering, Cairo University, Giza 12613, Egypt.
authors.forename: Ahmed A
authors.initials: AA
authors.lastname: Gomaa
authors.affiliation: Department of Food, Bioprocessing and Nutrition Sciences, North Carolina State University, Raleigh, NC 27695, USA.
authors.forename: Alexandra E
authors.initials: AE
authors.lastname: Briner
authors.affiliation: Department of Food, Bioprocessing and Nutrition Sciences, North Carolina State University, Raleigh, NC 27695, USA. Electronic address: rbarran@ncsu.edu.
authors.forename: Rodolphe
authors.initials: R
authors.lastname: Barrangou
authors.affiliation: Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, NC 27695, USA. Electronic address: cbeisel@ncsu.edu.
authors.forename: Chase L
authors.initials: CL
authors.lastname: Beisel
mesh_terms: D001407:Bacillus / Q000737:chemistry / Q000378:metabolism; D064130:CRISPR-Associated Proteins / Q000737:chemistry* / Q000235:genetics*; D064113:CRISPR-Cas Systems; D004926:Escherichia coli / Q000737:chemistry / Q000378:metabolism; D005603:Francisella / Q000737:chemistry / Q000378:metabolism; D057166:High-Throughput Screening Assays / Q000379:methods*; D017434:Protein Structure, Tertiary; D048249:Streptococcus thermophilus / Q000737:chemistry / Q000378:metabolism
chemical_list: D064130:CRISPR-Associated Proteins