Anti-CRISPR protein reveals sgRNA vulnerability in Cas9

Bacteriophages evolve anti-CRISPR proteins to evade bacterial immune defences, with over 120 families identified targeting different CRISPR-Cas systems through varied mechanisms. Most anti-Cas9 proteins achieve broad inhibition by targeting conserved protein domains, such as HNH or RuvC nucleases, but the mechanism by which AcrIIA27 blocks both type II-A and II-C Cas9 orthologs has remained unclear. To investigate this, researchers determined the cryo-electron microscopy structure of SpyCas9-sgRNA-AcrIIA27 at 2.68 Å resolution, revealing that AcrIIA27 uniquely binds the negatively charged phosphate backbone of solvent-exposed PAM-proximal sgRNA through positively charged loops, positioning near the PAM-binding pocket to sterically block DNA substrate recognition.

Biochemical assays confirmed that mutations disrupting these RNA contacts abolished inhibitory activity against SpyCas9, SauCas9, and Nme1Cas9. This mechanism suggested that solvent-exposed guide RNA regions might generally impede editing by enabling nonspecific binding of cellular proteins. Testing this hypothesis, the researchers truncated analogous PAM-proximal or transposon-adjacent motif-proximal RNA regions in SauCas9, FrCas9, and IS607 TnpB systems, achieving 1.2- to 16.2-fold improvements in genome-editing efficiency across multiple human cell loci whilst maintaining or improving specificity.

The study was led by Zhiwei Huang from the Harbin Institute of Technology in China. It was published in Nature Structural & Molecular Biology on 19 January 2026.