A positive, growth-based PAM screen identifies noncanonical motifs recognized by the <i>S. pyogenes</i> Cas9
Daphne Collias, Ryan T. Leenay, Rebecca Slotkowski, Zhicheng Zuo, Scott P. Collins, B. A. McGirr, Jin Liu, Chase L. Beisel
Abstract
. 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.