NmeCas9 is an intrinsically high-fidelity genome-editing platform

Abstract

BackgroundThe development of CRISPR 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, wildtype 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 to achieve single-site accuracy within large genomes. In addition to its off-targeting propensity, SpyCas9 is encoded by a relatively large open reading frame, limiting its utility in applications that require size-restricted delivery strategies such as adeno-associated virus vectors. In contrast, some genome-editing-validated Cas9 orthologs are considerably smaller and therefore better suited for viral delivery.ResultsHere we show that wildtype NmeCas9, when programmed with guide sequences of the natural length of 24 nucleotides, exhibits a nearly complete absence of unintended editing in human cells, even when targeting sites that are prone to off-target activity with wildtype 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.ConclusionsOur 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.Electronic supplementary materialThe online version of this article (10.1186/s13059-018-1591-1) contains supplementary material, which is available to authorized users.

Tissue-restricted genome editing in vivo specified by microRNA-repressible anti-CRISPR proteins

“…3B), and the SpyCas9 target site STS3 in the VEGFA gene ( Fig. 3C; Amrani et al 2018;Edraki et al 2018). In HEK293T cells, AcrIIC3 and AcrIIA4 robustly inhibited genome editing by Nme1/ 2Cas9 and SpyCas9, respectively, as expected ( Fig.…”

Improving CRISPR-Cas9 Genome Editing Efficiency by Fusion with Chromatin-Modulating Peptides

“…17 The requirement for a unique PAM by the nuclease is potentially useful for certain applications, and it has also been suggested that Cas9 nucleases with requirement for a long PAM might be naturally more specific. 19,20 To test whether we can enhance the activity of this smaller nuclease by the Cas9-CMP fusion strategy, we constructed the SpaCas9HN1HB1, Spa-Cas9HN1H1G, and SpaCas9HN1CHD1 fusion nucleases (Supplementary File S1). We also modified the previously described sgRNA scaffold by introducing a U to A mutation on the crRNA moiety to disrupt a track of five consecutive Us and a corresponding A to U mutation on the tracrRNA moiety to maintain the A-U base pairing (Supplementary File S1).…”

“…On the other hand, with >200 Cas9 orthologs predicted from the bacterial genome sequencing data, 15 it is a viable alternative to explore Cas9 diversity to increase targeting density. Indeed, several Cas9 orthologs with distinct PAM requirements have been identified, [16][17][18][19][20][21][22] but the majority of these orthologs appear to be more susceptible than SpyCas9 to target-to-target variation in cellular environments, limiting their widespread adoption.…”

Computational design of anti-CRISPR proteins with improved inhibition potency and expanded specificity

“…AcrIIC1 chimeras were created by Golden Gate cloning as follows: the plasmid encoding AcrIIC1 was first linearized at a selected position in the AcrIIC1 coding sequence via around-the-horn PCR; LOV2-, PDZ-and mCherrycoding sequences were then amplified by matching primers introducing optional GS linker encoding sequences and ligated into the linearized vector backbone; point mutations and protein tags were introduced via around-the-horn PCR via the primer overhangs. Annealed oligonucleotides corresponding to the target site sequence were cloned into the hybrid Cas9-sgRNA vectors via SapI (NmeCas9) or BsaI (SauCas9) restriction sites as described previously 20,34 . Supplementary Fig.…”