Journal Article

NmeCas9 is an intrinsically high-fidelity genome-editing platform

Amrani, Nadia, Fuller, Chris K., Sasaki, Kanae E., Fazzio, Thomas G., Zhu, Lihua Julie, Cameron, Peter, Ibraheim, Raed, Mir, Aamir, Sontheimer, Erik J., Edraki, Alireza, Gupta, Ankit, Liu, Pengpeng, Settle, Alexander H., Wolfe, Scot A., Wu, Tong, Gao, Xin D., McGovern, Kyle, Lied, Alexandra M., Donohoue, Paul D.

2018

Overview

Background The 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. Results Here 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′-N 4 GATT-3′), for NmeCas9 genome editing in human cells. Conclusions 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.

• Publisher: BioMed Central,Springer Nature B.V,BMC
• Subject: Amino acids / Animal Genetics and Genomics / Animals / Bioinformatics / Biomedical and Life Sciences / biomedical research / Cas9 / CRISPR / CRISPR-associated endonuclease 9 / CRISPR-Associated Protein 9 - metabolism / Deoxyribonucleic acid / Dependoparvovirus / DNA / Enzyme kinetics / Evolutionary Biology / Fidelity / gene editing / Gene Editing - methods / genome / Genomes / Human Genetics / Humans / Insights from Genome Editing / Life Sciences / Microbial Genetics and Genomics / Neisseria meningitidis / Neisseria meningitidis - enzymology / nucleotides / Off-target / Plant Genetics and Genomics / Proteins / Protospacer adjacent motif / Ribonucleic acid / RNA / sgRNA / Streptococcus pyogenes