9LKJ

Structure of Cas9-sgRNA-A27

Summary for 9LKJ

• Entry DOI: 10.2210/pdb9lkj/pdb
• EMDB information: 63180
• Descriptor: Phage protein, CRISPR-associated endonuclease Cas9/Csn1, RNA (98-MER) (3 entities in total)
• Functional Keywords: host defence, immune system
• Biological source: Streptococcus pyogenes; More
• Total number of polymer chains: 3
• Total formula weight: 197973.38

Authors

Yu, L.,Zhu, Y.,Lu, Z.,Huang, Z. (deposition date: 2025-01-16, release date: 2026-01-21, Last modification date: 2026-08-05)

Primary citation

Yu, L.,Yin, M.,Zhu, Y.,Lu, Z.,Xiao, B.,Zhou, F.,Yu, Y.,Huang, Z.
An anti-CRISPR targets the sgRNA to block Cas9 and guides the design of enhanced genome editors.
Nat.Struct.Mol.Biol., 33:318-329, 2026

PubMed Abstract: Bacteriophages have evolved anti-CRISPR (Acr) proteins to combat the adaptive immunity provided by bacterial CRISPR-Cas systems. Here, we report the cryo-electron microscopy structure of an anti-Cas9 protein AcrIIA27 bound to SpyCas9-sgRNA (single guide RNA) complex. Our structure reveals that AcrIIA27 binds the solvent-exposed phosphate backbone of the sgRNA, acting as a potent inhibitor of diverse Cas9 orthologs. AcrIIA27 in the structure is positioned near the protospacer-adjacent motif DNA-binding pocket on SpyCas9, causing steric hindrance that prevents substrate DNA recognition. This mechanism suggests solvent-exposed regions of sgRNAs (PTP RNAs), prone to nonspecific binding of positively charged components, may compromise CRISPR-Cas genome-editing efficiency. Indeed, truncations of the PTP RNAs in different editing systems significantly enhance genome-editing efficiency in human cells. Overall, our findings reveal a previously uncharacterized inhibition mechanism of an anti-Cas protein and offers a general strategy for developing more efficient genome-editing tools.

PubMed: 41555077
DOI: 10.1038/s41594-025-01741-z

Experimental method

ELECTRON MICROSCOPY (2.68 Å)