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<p><i>Staphylococcus aureus</i> Cas9 (SaCas9) is smaller than the widely used <i>Streptococcus pyogenes</i> 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.</p>

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

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年4月16日

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

摘要

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.