UMass Chan Medical School Faculty Publications

UMMS Affiliation

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

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Article Preprint


Amino Acids, Peptides, and Proteins | Cells | Enzymes and Coenzymes | Genetic Phenomena | Genetics and Genomics | Molecular Biology | Nucleic Acids, Nucleotides, and Nucleosides


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.


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

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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.

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bioRxiv 172650; doi: Link to preprint on bioRxiv service.


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

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