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

Most known anti-CRISPR proteins exert inhibition via solitary functional pathways, and few natural Acr homologs have been reported to possess coupled inhibitory mechanisms. Here, we demonstrate that AcrIIC1Boe represses Nme1Cas9 activity through loop-coupled inhibitory effects. Beyond canonical occlusion of the HNH catalytic site, the unique extended loop of AcrIIC1Boe further perturbs sgRNA binding and restricts R-loop maturation to reinforce inhibition. Strikingly, loop deletion completely abrogated this layered inhibitory capacity, confirming that the loop is indispensable for full anti-CRISPR function. Consistently, transplantation of this functional loop into the weakly active AcrIIC1Nme1 markedly upgraded its inhibitory performance. Our findings reveal an unprecedented loop-governed coupled inhibition mode, clarify the structural basis for functional divergence within the AcrIIC1 family, and provide a modular engineering strategy for the rational design and optimization of high-potency anti-CRISPR proteins.