Anti-CRISPR proteins in Gluconobacter oxydans inactivate FnCas12a by acetylation

Gluconobacter oxydans is an important chassis cell for one-step production of vitamin C. Previous studies reported that CRISPR/Cas12a is naturally inactivated in G. oxydans, but the specific mechanism remains unclear. Here, we identified anti-CRISPR proteins AcrVA6, AcrVA7 and AcrVA8 in G. oxydans. They functioned as acetyltransferases to inactivate FnCas12a by respectively acetylating Lys671, Lys589 and Lys823 of FnCas12a. Lys671 and Lys823 were related residues that recognise the protospacer-adjacent motif, modification of AcrVA6 and AcrVA8 untangled the interaction between FnCas12a and dsDNA, while Lys589 played an important role in binding to the crRNA-target DNA heteroduplex, AcrVA7 prevented the formation of FnCas12a-crRNA binary complexes. In addition, histone deacetylase HDAC11 was found to prevent modification of FnCas12a by AcrVA6. Quantum mechanical calculations showed that ser37 of AcrVA6, as an intermediate between acetyl group and receptor protein, achieves acetylation through ping-pong transfer mechanism. Finally, the acetyltransferase AcrVA6 and the deacetylase HDAC11 served as photoswitches by writing and erasing acetyl groups, respectively, to achieve continuous on-off of FnCas12a. Our study reveals different mechanisms by which acetyltransferase inactivates Cas12a and successfully applies reversible acetylation to the regulation of gene editing tools, providing new insights into the function and application of acetylation.