NmeCas9 is an intrinsically high-fidelity genome-editing platform

Genome Biology

Chris K. Fuller¹  Alexandra M. Lied¹  Paul D. Donohoue¹  Kyle McGovern¹  Alexander H. Settle¹  Peter Cameron¹  Pengpeng Liu²  Ankit Gupta²  Scot A. Wolfe²  Tong Wu²  Lihua Julie Zhu³  Thomas G. Fazzio³  Alireza Edraki⁴  Nadia Amrani⁴  Raed Ibraheim⁴  Aamir Mir⁴  Kanae E. Sasaki⁴  Erik J. Sontheimer⁴  Xin D. Gao⁴ 

[1] Caribou Biosciences, Inc.;Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School;Program in Molecular Medicine, University of Massachusetts Medical School;RNA Therapeutics Institute, University of Massachusetts Medical School;

关键词: Cas9;    CRISPR;    sgRNA;    Protospacer adjacent motif;    Off-target;    Neisseria meningitidis;   

DOI  :  10.1186/s13059-018-1591-1

来源: DOAJ

【 摘 要 】

Abstract 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′-N4GATT-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.

【 授权许可】

Unknown