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.