Type II CRISPR systems defend against invasive DNA by using Cas9 as an RNA-guided
nuclease that creates double-stranded DNA breaks. Dual RNAs (CRISPR RNA [crRNA] and
tracrRNA) are required for Cas9's targeting activities observed to date. Targeting requires a
protospacer adjacent motif (PAM) and crRNA-DNA complementarity. Cas9 orthologs
(including Neisseria meningitidis Cas9 [NmeCas9]) have also been adopted for genome
engineering. Here we examine the DNA cleavage activities and substrate requirements of …

Many strains of the human pathogen Neisseria meningitidis carry a compact Cas9
(NmeCas9) that can serve to limit genetic exchange via natural transformation. Cas9
orthologues (including NmeCas9) have recently been adopted for RNA-guided genome
engineering and DNA binding, adding to the need to define better their activities and
properties. The present invention examines DNA cleavage activities and substrate
requirements of NmeCas9, including a set of unusually complex PAM recognition patterns …