How the CRISPR Cas9 enzyme searches and recognizes DNA

Both binding and cleavage of DNA by Cas9-RNA require recognition of a short trinucleotide protospacer adjacent motif (PAM).

• Non-target DNA sequences that lack a nearby PAM are ignored by Cas9-RNA.
• DNA binding affinity increases with PAM density.
• Competition assays indicate that DNA strand separation and RNA-DNA pairing start at the PAM and move towards the target sequence's end.
• PAM interactions activate Cas9's ability to cut double-stranded DNA.
• Cas9 uses PAM recognition to efficiently locate potential target sites within large DNA molecules.

Simplified