Browsing Division of RNA biology of bacterial infections ([HIRI] RABI) by Authors
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; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany. (AAAS, 2020-07-15)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.