Title

NmeCas9 is an intrinsically high-fidelity genome editing platform [preprint]

Authors

Nadia Amrani, University of Massachusetts Medical SchoolFollow
Xin D. Gao, University of Massachusetts Medical School
Pengpeng Liu, University of Massachusetts Medical SchoolFollow
Alireza Edraki, University of Massachusetts Medical School
Aamir Mir, University of Massachusetts Medical School
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; Program in Molecular Medicine; Department of Molecular, Cell and Cancer Biology; Department of Biochemistry and Molecular Pharmacology

Publication Date

2018-05-09

Document Type

Article Preprint

Disciplines

Amino Acids, Peptides, and Proteins | Cells | Enzymes and Coenzymes | Genetic Phenomena | Genetics and Genomics | Molecular Biology | 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, wild-type 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 (e.g. mammalian) genomes. In addition to its off-targeting propensity, SpyCas9 is encoded by a relatively large (~4.2 kb) 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 (e.g. from Staphylococcus aureus, Campylobacter jejuni, Geobacillus stearothermophilus and Neisseria meningitidis) are considerably smaller and therefore better suited for viral delivery.

Results: Here we show that wild-type NmeCas9, when programmed with guide sequences of natural length (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

molecular biology, NmeCas9, genome editing, Streptococcus pyogenes SF370, SpyCas9, Cas9, CRISPR, sgRNA, Protospacer adjacent motif, off-target, Neisseria meningitidis

Rights and Permissions

The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license.

DOI of Published Version

10.1101/172650

Source

bioRxiv 172650; doi: https://doi.org/10.1101/172650. Link to preprint on bioRxiv service.

Comments

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

Journal/Book/Conference Title

bioRxiv

Repository Citation

Amrani N, Gao XD, P, Edraki A, Mir A, Ibraheim R, Gupta A, Sasaki KE, Wu T, Fazzio TG, Zhu LJ, Wolfe SA, Sontheimer EJ. (2018). NmeCas9 is an intrinsically high-fidelity genome editing platform [preprint]. UMass Chan Medical School Faculty Publications. https://doi.org/10.1101/172650. Retrieved from https://escholarship.umassmed.edu/faculty_pubs/1559

Creative Commons License

This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 4.0 License.