AcrVA3 Is a Double Strand DNA ‐Cleaving Anti‐ CRISPR That Indirectly Inhibits Cas12

CRISPR‐Cas12 systems protect bacteria from foreign DNA, but are themselves targeted by anti‐CRISPR (Acr) proteins evolved by phages. Among the eight known AcrV proteins that inhibit Cas12‐based type V CRISPR‐Cas systems, the mechanisms of all except AcrVA3 have been structurally and biochemically characterized. Here, we report the high‐resolution structure of AcrVA3 and examine its inhibitory function in vitro. Unexpectedly, AcrVA3 does not directly work on Cas12. Instead, it exhibits double‐stranded DNA (dsDNA) cleavage activity, suggesting an indirect mechanism of CRISPR inhibition through DNA degradation. This unique DNA‐centric strategy contrasts with previously known Acr mechanisms and expands our understanding of how mobile genetic elements evade CRISPR immunity.