8ZCZ

Cryo-EM structure of eSaCas9_NNG-guide RNA-target DNA complex in an intermediate state

Summary for 8ZCZ

• Entry DOI: 10.2210/pdb8zcz/pdb
• EMDB information: 39942
• Descriptor: CRISPR-associated endonuclease Cas9, sgRNA, Target DNA strand, ... (4 entities in total)
• Functional Keywords: crispr-cas9, genome engineering, hydrolase-rna-dna complex, dna binding protein
• Biological source: Staphylococcus aureus; More
• Total number of polymer chains: 4
• Total formula weight: 182899.76

Authors

Omura, S.N.,Nakagawa, R.,Yamashita, K.,Nishimasu, H.,Nureki, O. (deposition date: 2024-04-30, release date: 2025-11-05, Last modification date: 2026-04-29)

Primary citation

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.
Engineering a compact high-fidelity Staphylococcus aureus Cas9 variant with broader targeting range and mechanistic insights into its activation.
Nat Commun, 17:-, 2026

PubMed 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.

PubMed: 41991526
DOI: 10.1038/s41467-026-71626-2

Experimental method

ELECTRON MICROSCOPY (2.9 Å)