Engineering a compact high-fidelity Staphylococcus aureus Cas9 variant with broader targeting range and mechanistic insights into its activation

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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Publication Details

Journal
Nature Communications
Published
2026-04-16
DOI
https://doi.org/10.1038/s41467-026-71626-2
Primary Topic
CRISPR and Genetic Engineering
Type
article
Field-Weighted Citation Impact
0.00

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article

Engineering a compact high-fidelity Staphylococcus aureus Cas9 variant with broader targeting range and mechanistic insights into its activation

Yuji Kashiwakura, Satoshi N. Omura, Hideto Mori, Tsukasa Ohmori et al.
Nature Communications
CRISPR and Genetic Engineering
article

Engineering a compact high-fidelity Staphylococcus aureus Cas9 variant with broader targeting range and mechanistic insights into its activation

Yuji Kashiwakura, Satoshi N. Omura, Hideto Mori, Tsukasa Ohmori, Osamu Nureki, Shohei Kajimoto, Nozomu Yachie, Hiroshi Nishimasu, Takafumi Hiramoto, Kasey Jividen, Soh Ishiguro, Keitaro Yamashita, Ryoya Nakagawa, Shengdar Q. Tsai, Kio Horinaka, Hisato Hirano, Mamoru Tanaka, Sae Okazaki, Kosuke Onishi
article en

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.

Nature CommunicationsVol. 17(1)
St. Jude Children's Research Hospital (US), University of British Columbia (CA), Jichi Medical University (JP), Tokyo University of Science (JP), Keio University (JP), Inamori Foundation (JP), The University of Tokyo (JP), The University of Osaka (JP)
Japan Agency for Medical Research and Development, Cabinet Office, Government of Japan, Jichi Medical University, University of Tokyo, National Institutes of Health, University of California, San Francisco, Japan Society for the Promotion of Science, National Institute of Allergy and Infectious Diseases, Core Research for Evolutional Science and Technology
Partnerships for the goals
Openalex Percentile: Top 24%
CRISPR and Genetic Engineering
DOIOpen Access PDFOpenAlex
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