Closely related type II-C Cas9 orthologs recognize diverse PAMs

Autosomal dominant diseases can be treated by allele-specific disruption of mutant alleles. If the missense mutation generates a novel protospacer-adjacent motif (PAM), CRISPR-Cas9 can distinguish mutant alleles from wild-type alleles by a PAM-specific approach. Therefore, it is crucial to develop a CRISPR toolbox capable of recognizing multiple PAMs. Here, by using a GFP-activation assay, we tested the activities of 29 type II-C orthologs closely related to Nme1Cas9, 25 of which are active in human cells. These orthologs recognize diverse PAMs with variable length and nucleotide preference, including purine-rich, pyrimidine-rich, and mixed purine and pyrimidine PAMs. We characterized in depth the activity and specificity of Nsp2Cas9. We also generated a chimeric Cas9 nuclease that recognizes a simple N₄C PAM, representing the most relaxed PAM preference for compact Cas9s to date. These Cas9 nucleases significantly enhance our ability to perform allele-specific genome editing.