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A positive, growth-based PAM screen identifies noncanonical motifs recognized by the S. pyogenes Cas9.
Collias, D Leenay, R T Slotkowski, R A Zuo, Z Collins, S P McGirr, B A Liu, J Beisel, C L
Collias, D
Leenay, R T
Slotkowski, R A
Zuo, Z
Collins, S P
McGirr, B A
Liu, J
Beisel, C L
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2020-07-15
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CRISPR technologies have overwhelmingly relied on the Streptococcus pyogenes Cas9 (SpyCas9), with its consensus NGG and less preferred NAG and NGA protospacer-adjacent motifs (PAMs). Here, we report that SpyCas9 also recognizes sequences within an N(A/C/T)GG motif. These sequences were identified on the basis of preferential enrichment in a growth-based screen in Escherichia coli. DNA binding, cleavage, and editing assays in bacteria and human cells validated recognition, with activities paralleling those for NAG(A/C/T) PAMs and dependent on the first two PAM positions. Molecular-dynamics simulations and plasmid-clearance assays with mismatch-intolerant variants supported induced-fit recognition of an extended PAM by SpyCas9 rather than recognition of NGG with a bulged R-loop. Last, the editing location for SpyCas9-derived base editors could be shifted by one nucleotide by selecting between (C/T)GG and adjacent N(C/T)GG PAMs. SpyCas9 and its enhanced variants thus recognize a larger repertoire of PAMs, with implications for precise editing, off-target predictions, and CRISPR-based immunity.
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Sci Adv. 2020 Jul 15;6(29):eabb4054. doi: 10.1126/sciadv.abb4054.
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en
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2375-2548
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Attribution-NonCommercial-ShareAlike 4.0 International