A positive, growth-based PAM screen identifies noncanonical motifs recognized by the <i>S. pyogenes</i> Cas9.
basic_science · Level V
Where this comes from
- Record sourced from PubMed, PMID 32832642.
- Also identified by DOI 10.1126/sciadv.abb4054 and PMC identifier 7439565.
- Licence recorded as CC BY-NC.
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Abstract
CRISPR technologies have overwhelmingly relied on the <i>Streptococcus pyogenes</i> 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 <i>Escherichia coli</i>. 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.