Structural Basis of Recognition of Anti‐ CRISPR Operon by Aca3

The CRISPR-Cas system equips bacteria with adaptive immunity by storing fragments of invading nucleic acids in CRISPR loci and deploying Cas proteins to recognize and degrade matching sequences. In turn, bacteriophages have evolved small anti-CRISPR (Acr) that neutralize diverse CRISPR-Cas types. Acr genes are often co-encoded with transcriptional regulators called anti-CRISPR-associated (Aca) proteins, which suppress acr expression. Although 13 Aca families have been identified through bioinformatic analysis, detailed information on their target DNA-binding mechanisms and the inhibition of acr expression remains limited. Here, we report the high-resolution structure of Aca3 and delineate its DNA-binding interface. We demonstrate that Aca3 selectively recognizes inverted repeats upstream of its cognate acr gene, AcrIIC1. Mutational analyses of key helix-turn-helix residues confirm their essential roles in promoter engagement. Together, these results reveal the molecular basis for Aca3-mediated control of anti-CRISPR expression and expand our understanding of regulatory strategies that phages employ to modulate host CRISPR-Cas immunity.