Structural basis for loop-dependent inhibition by AcrIIC1 anti-CRISPR proteins

Most characterized anti-CRISPR proteins act via well-defined core inhibitory mechanisms, yet how auxiliary structural elements modulate and potentiate these canonical processes remains poorly understood. Here, we show that AcrIIC1Boe represses Nme1Cas9 activity through an integrated, loop-coupled regulatory mode. Based on canonical occlusion of the HNH catalytic site, the extended loop of AcrIIC1Boe provides an additional coupled regulatory layer to remodel sgRNA spacer arrangement and R-loop progression, thereby enhancing Cas9 inhibition. Loop deletion largely abrogates robust inhibition, indicating that this loop module is indispensable for full AcrIIC1 inhibitory potency. Consistently, transplantation of this functional loop into the weakly active AcrIIC1Nme1 efficiently elevates its inhibitory performance. Our findings identify a loop-mediated regulatory strategy that potentiates the canonical HNH occlusion mechanism, clarify the structural basis for functional divergence across AcrIIC1 homologs, and offer a modular engineering principle for the rational design and optimization of anti-CRISPR proteins with enhanced potency. Anti-CRISPR proteins inhibit bacterial CRISPR immunity, but how auxiliary structural elements enhance inhibition is unclear. Here, authors show that an extended AcrIIC1 loop supports HNH-domain engagement and modulates sgRNA positioning and R-loop formation to strengthen Cas9 inhibition.