TY - JOUR
T1 - Identifying and Visualizing Functional PAM Diversity across CRISPR-Cas Systems
AU - Leenay, Ryan T.
AU - Maksimchuk, Kenneth R.
AU - Slotkowski, Rebecca A.
AU - Agrawal, Roma N.
AU - Gomaa, Ahmed A.
AU - Briner, Alexandra E.
AU - Barrangou, Rodolphe
AU - Beisel, Chase L.
N1 - Funding Information:
We thank Stacie Meaux at Research Square for developing the video summary contained in Movie S1 , Brooke McGirr for assistance with cloning and recombineering, and Michelle Luo for devising the PAM-SCANR acronym and for critical reading of the manuscript. We also thank Sarah Schuett at the NCSU CVM Cell Sorting Facility for all FACS work. The BhaloCascade plasmid was a gift from Ailong Ke, and the SpydCas9 plasmid was a gift from Lei Qi (Addgene #44249). The work was supported by funding from the National Science Foundation ( CBET-1403135 to C.L.B. and R.B., MCB-1452902 to C.L.B.), the Kenan Institute of Engineering, Technology and Science (to C.L.B.), the National Institutes of Health ( 5T32GM008776-15 to R.T.L.), and an NCSU undergraduate research grant (to R.A.S.). R.B. is a shareholder and advisor of Caribou Biosciences and a founder of Intellia Therapeutics and a member on its scientific advisory board. A.A.G. and C.L.B. are founders of Locus Biosciences and members of its scientific advisory board.
Publisher Copyright:
© 2016 Elsevier Inc.
PY - 2016/4/7
Y1 - 2016/4/7
N2 - 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.
AB - 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.
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U2 - 10.1016/j.molcel.2016.02.031
DO - 10.1016/j.molcel.2016.02.031
M3 - Article
C2 - 27041224
AN - SCOPUS:84979464834
SN - 1097-2765
VL - 62
SP - 137
EP - 147
JO - Molecular Cell
JF - Molecular Cell
IS - 1
ER -