Engineering a compact high-fidelity Staphylococcus aureus Cas9 variant with broader targeting range and mechanistic insights into its activation
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Abstract
Staphylococcus aureus Cas9 (SaCas9) is smaller than the widely used Streptococcus pyogenes Cas9 (SpCas9) and has been harnessed for gene therapy using an adeno-associated virus vector. However, SaCas9 requires a longer NNGRRT (where N is any nucleotide and R is A or G) protospacer adjacent motif (PAM) for target DNA recognition, thereby restricting the targeting range. Although PAM-relaxed Cas9 variants have been developed, expanded targeting is often accompanied by compromised target specificity. Here, we report the rational engineering of eSaCas9-NNG, a SaCas9 variant that recognizes relaxed NNG PAMs while maintaining high target fidelity, thereby overcoming a fundamental trade-off in Cas9-based genome editing. eSaCas9-NNG efficiently induces indels and base conversions at endogenous sites bearing NNG PAMs in human cells and mice, with editing efficiencies comparable to those of other PAM-relaxed nucleases, including SpRY, SpG, and iGeoCas9, but with reduced off-target activity. We further determine the cryo-electron microscopy structures of eSaCas9-NNG in five distinct functional states, revealing the structural basis for its relaxed PAM recognition, improved target specificity, and nuclease activation. Overall, our findings demonstrate that eSaCas9-NNG could be used as a versatile genome editing tool for in vivo gene therapy, and improve our mechanistic understanding of the diverse CRISPR-Cas9 nucleases.
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- Authors
- Omura, S. N., Nakagawa, R., Kajimoto, S., Okazaki, S., Ishiguro, S., Mori, H., Onishi, K., Kashiwakura, Y., Hiramoto, T., Horinaka, K., Tanaka, M., Hirano, H., Jividen, K., Yamashita, K., Tsai, S. Q., Yachie, N., Ohmori, T., Nishimasu, H., & Nureki, O.
- Affiliations
- The University of Tokyo (JP)
- Research Center for Advanced Science and Technology, The University of Tokyo (JP)
- University of British Columbia (CA)
- Keio University (JP)
- Jichi Medical University (JP)
- St. Jude Children's Research Hospital (US)
- Corresponding author
- Nishimasu, Hiroshi
- Nureki, Osamu
- ORCID
- Venue
- Nature Communications
- Published
- 2026-04-16
- Volume
- 17
- Issue
- 1
- ISSN
- 2041-1723 (ISSN-L)
- OpenAlex
- W7154623531
- Cited by
- 2
- References
- 67
- Citations by year
- 2026: 2
- FWCI
- 4.13
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- Locations
- Nature Communications (publishedVersion, cc-by-nc-nd)
- Research Square (acceptedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by-nc-nd)
- PubMed (publishedVersion)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
- Funders
- Japan Agency for Medical Research and Development (JP25ama121012)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA regulation and disease
- Health Sciences › Medicine › Oncology › CAR-T cell therapy research
- Keywords
- SaCas9, genome editing, off-target effects, nuclease activation, adeno-associated virus gene therapy, in vivo gene therapy, base conversion
- MeSH terms
- Gene Editing, CRISPR-Associated Protein 9, Animals, Bacterial Proteins, Humans, Staphylococcus aureus, Streptococcus pyogenes, Protein Engineering, Cryoelectron Microscopy, Mice, HEK293 Cells, CRISPR-Cas Systems
About these checks
Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.
Statistics and arithmetic are recomputed, not judged.
Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.
What counts as a claim ›How we measure confidence ›How we check this ›
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