Structural basis for dual mechanism of Cas2/3 nuclease inhibition by anti-CRISPR protein AcrIF19

Abstract CRISPR-Cas systems are prokaryotic immune mechanisms often targeted by phage-encoded anti-CRISPR (Acr) proteins. This study characterizes AcrIF19, a potent inhibitor of the type I-F system in Pectobacterium atrosepticum . The cryo-EM structure of the apo Cas2/3 and Cas2/3-AcrIF19 complex reveals a dual inhibitory mechanism. AcrIF19 employs a negatively charged β 2 -β 3 loop to sterically occlude the non-target DNA strand entry channel, acting as a competitive inhibitor to disrupt Cas2/3 recruitment. Concurrently, this steric occlusion impedes ssDNA-mediated allosteric activation, which locks the critical helix-like loop motif in an inhibitory conformation and thereby abrogates DNA cleavage activity. AcrIF19 represents an anti-CRISPR protein inhibiting Cas2/3 via two different mechanisms, integrating a competitive ssDNA inhibitor with an allosteric blockade to suppress both target recruitment and DNA cleavage.