An anti-CRISPR protein disables type V Cas12a by acetylation

Phages use anti-CRISPR proteins to deactivate the CRISPR–Cas system. The mechanisms for the inhibition of type I and type II systems by anti-CRISPRs have been elucidated. However, it has remained unknown how the type V CRISPR–Cas12a (Cpf1) system is inhibited by anti-CRISPRs. Here we identify the anti-CRISPR protein AcrVA5 and report the mechanisms by which it inhibits CRISPR–Cas12a. Our structural and biochemical data show that AcrVA5 functions as an acetyltransferase to modify Moraxella bovoculi (Mb) Cas12a at Lys635, a residue that is required for recognition of the protospacer-adjacent motif. The AcrVA5-mediated modification of MbCas12a results in complete loss of double-stranded DNA (dsDNA)-cleavage activity. In contrast, the Lys635Arg mutation renders MbCas12a completely insensitive to inhibition by AcrVA5. A cryo-EM structure of the AcrVA5-acetylated MbCas12a reveals that Lys635 acetylation provides sufficient steric hindrance to prevent dsDNA substrates from binding to the Cas protein. Our study reveals an unprecedented mechanism of CRISPR–Cas inhibition and suggests an evolutionary arms race between phages and bacteria. Zhiwei Huang and colleagues report structural and biochemical data showing that the anti-CRISPR protein AcrVA5 functions as an acetyltransferase, modifying MbCas12a at Lys635, a residue required for PAM recognition. Acetylation of Lys635 creates a steric clash that prevents binding of target DNA.