A positive, growth-based PAM screen identifies noncanonical motifs recognized by the S. pyogenes Cas9.
Daphne ColliasR T LeenayRebecca A SlotkowskiZ ZuoS P CollinsB A McGirrJin LiuChase L BeiselPublished in: Science advances (2020)
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.
Keyphrases
- crispr cas
- genome editing
- dna binding
- molecular dynamics simulations
- high throughput
- escherichia coli
- transcription factor
- copy number
- genome wide
- biofilm formation
- molecular docking
- high glucose
- gene expression
- diabetic rats
- oxidative stress
- single cell
- atopic dermatitis
- staphylococcus aureus
- cystic fibrosis
- pseudomonas aeruginosa
- klebsiella pneumoniae