Translation-dependent downregulation of Cas12a mRNA by an anti-CRISPR protein

Bacterial CRISPR-Cas systems cleave the genomic DNA of bacteriophage and other mobile genetic elements. Many temperate phages encode anti-CRISPR (Acr) proteins that use diverse and innovative mechanisms to inhibit CRISPR-Cas and stabilize lysogeny. We previously reported that a Moraxella bovoculi prophage encodes two anti-CRISPR proteins, AcrVA1 and AcrVA2, that inhibit MbCas12a. Whereas AcrVA1 inactivates CRISPR-Cas12a complexes by cleaving the CRISPR RNA (crRNA), the mechanism and utility of AcrVA2 have remained unclear. Here, we show that AcrVA2 unexpectedly inhibits Cas12a biogenesis by binding to the nascent Cas12a polypeptide and triggering degradation of its mRNA. Mutations in the first 15 amino acids of Cas12a abolish binding, downregulation, and inhibition by AcrVA2, while altering the Cas12a codon sequence and promoter has no effect. These co-encoded anti-CRISPRs therefore use complementary strategies to recognize conserved features in Cas12a and inhibit its DNA targeting and biogenesis. Beyond Moraxella, AcrVA2 is broadly distributed across numerous bacterial clades, including where Cas12a systems are not found, suggesting that this novel mechanism of gene regulation may target other proteins. ### Competing Interest Statement UCSF has filed a patent on the use of inhibitors of CRISPR-Cas12a, on which NDM and JBD are listed as inventors.