Identification of SaCas9 orthologs containing a conserved serine residue that determines simple NNGG PAM recognition

AU Due: to Pleaseconfirmthatallheadinglevelsarerepresentedcorrectly different nucleotide preferences at target sites, no single : Cas9 is capable of editing all sequences. Thus, this highlights the need to establish a Cas9 repertoire covering all sequences for efficient genome editing. Cas9s with simple protospacer adjacent motif (PAM) requirements are particularly attractive to allow for a wide range of genome editing, but identification of such Cas9s from thousands of Cas9s in the public database is a challenge. We previously identified PAMs for 16 SaCas9 orthologs. Here, we compared the PAM-interacting (PI) domains in these orthologs and found that the serine residue corresponding to SaCas9 N986 was associated with the simple NNGG PAM requirement. Based on this discovery, we identified five additional SaCas9 orthologs that recognize the NNGG PAM. We further identified three amino acids that determined the NNGG PAM requirement of SaCas9. Finally, we engineered Sha2Cas9 and SpeCas9 to generate high-fidelity versions of Cas9s. Importantly, these natural and engineered Cas9s displayed high activities and distinct nucleotide preferences. Our study offers a new perspective to identify SaCas9 orthologs with NNGG PAM requirements, expanding the Cas9 repertoire.