ABSTRACT
The development of CRISPR-Cas9 RNA-guided genome editing has transformed biomedical research. Most applications reported thus far rely upon the Cas9 protein from Streptococcus pyogenes SF370 (SpyCas9). With many RNA guides, SpyCas9 can induce significant levels of unintended mutations at near-cognate sites, necessitating substantial efforts toward the development of strategies to minimize off-target activity. Although the genome-editing potential of thousands of other Cas9 orthologs remains largely untapped, it is not known how many will require similarly extensive engineering efforts to achieve single-site accuracy within large (e.g. mammalian) genomes. In addition to its off-targeting propensity, SpyCas9 is encoded by a relatively large (∼4.2 kb) open reading frame (ORF), limiting its utility in applications that require size-restricted delivery strategies such as adeno-associated virus (AAV) vectors. In contrast, some genome-editing-validated Cas9 orthologs [e.g. Staphylococcus aureus Cas9 (SauCas9), Campylobacter jejuni Cas9 (CjeCas9), and Neisseria meningitidis Cas9 (NmeCas9)] are considerably smaller and therefore better suited for viral delivery. Here we show that wild-type NmeCas9, when programmed with guide sequences of natural length (24 nucleotides), exhibits a nearly complete absence of unintended targeting in human cells, even when targeting sites that are highly prone to off-target activity when employing SpyCas9. We also validate at least six variant protospacer adjacent motifs (PAMs), in addition to the preferred consensus PAM (5’-N4GATT-3’), for NmeCas9 genome editing in human cells. Our results show that NmeCas9 is a naturally high-fidelity genome editing enzyme, and suggest that additional Cas9 orthologs may prove to exhibit similarly high accuracy, even without extensive engineering efforts.
