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Title

NmeCas9 is an intrinsically high-fidelity genome-editing platform

Authors

Nadia Amrani, University of Massachusetts Medical SchoolFollow
Xin D. Gao, University of Massachusetts Medical SchoolFollow
Pengpeng Liu, University of Massachusetts Medical SchoolFollow
Alireza Edraki, University of Massachusetts Medical SchoolFollow
Aamir Mir, University of Massachusetts Medical SchoolFollow
Raed Ibraheim, University of Massachusetts Medical SchoolFollow
Ankit Gupta, University of Massachusetts Medical SchoolFollow
Kanae E. Sasaki, University of Massachusetts Medical School
Tong Wu, University of Massachusetts Medical SchoolFollow
Thomas G. Fazzio, University of Massachusetts Medical SchoolFollow
Lihua Julie Zhu, University of Massachusetts Medical SchoolFollow
Scot A. Wolfe, University of Massachusetts Medical SchoolFollow
Erik J. Sontheimer, University of Massachusetts Medical SchoolFollow

UMMS Affiliation

RNA Therapeutics Institute; Department of Molecular, Cell and Cancer Biology; Department of Biochemistry and Molecular Pharmacology; Program in Molecular Medicine; Graduate School of Biomedical Sciences

Publication Date

2018-12-05

Document Type

Article

Disciplines

Bioinformatics | Computational Biology | Genetic Phenomena | Genomics | Nucleic Acids, Nucleotides, and Nucleosides

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.

Keywords

CRISPR, Cas9, Neisseria meningitidis, Off-target, Protospacer adjacent motif, sgRNA

Rights and Permissions

© The Author(s). 2018 Open Access: This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

DOI of Published Version

10.1186/s13059-018-1591-1

Source

Genome Biol. 2018 Dec 5;19(1):214. doi: 10.1186/s13059-018-1591-1. Link to article on publisher's site

Journal/Book/Conference Title

Genome biology

Comments

Full author list omitted for brevity. For the full list of authors, see article.

Related Resources

Link to Article in PubMed

PubMed ID

30518407

Repository Citation

Amrani, Nadia; Gao, Xin D.; Pengpeng Liu; Edraki, Alireza; Mir, Aamir; Ibraheim, Raed; Gupta, Ankit; Sasaki, Kanae E.; Wu, Tong; Fazzio, Thomas G.; Zhu, Lihua Julie; Wolfe, Scot A.; and Sontheimer, Erik J., "NmeCas9 is an intrinsically high-fidelity genome-editing platform" (2018). Open Access Articles. 3702.
https://escholarship.umassmed.edu/oapubs/3702

Creative Commons License

This work is licensed under a Creative Commons Attribution 4.0 License.