Structural insights into Cas9 inhibition by AcrIIA17 via bridge helix interaction.
Anti-CRISPR (Acr) proteins have evolved in bacteriophages and mobile genetic elements to counteract CRISPR-Cas immune systems through diverse inhibitory mechanisms. Here, we present the crystal structure of AcrIIA17 and elucidate its mechanism of Staphylococcus aureus Cas9 (SauCas9) inhibition. AcrIIA17 adopts a previously uncharacterized protein fold and exists as a monomer in solution. Biochemical analyses reveal that AcrIIA17 inhibits SauCas9 activity in a strictly order-dependent manner, effectively suppressing DNA cleavage only when it engages Cas9 prior to single guide RNA (sgRNA) loading, whereas pre-assembled Cas9-sgRNA ribonucleoprotein (RNP) complexes are resistant to inhibition. Domain-mapping experiments demonstrate that AcrIIA17 directly binds to the bridge helix (BH) domain of SauCas9, and structure-guided mutagenesis confirms that this interaction is essential for its inhibitory function. Together, our findings identify AcrIIA17 as an Acr protein that targets the Cas9 BH domain and reveal the BH domain as a regulatory checkpoint in Cas9 activation.