AcrIIA7 hijacks tracrRNA to block CRISPR-Cas system.

The CRISPR-Cas9 system provides adaptive immunity against invading genetic elements through a dual-RNA-guided DNA cleavage mechanism. This system relies on the precise assembly of a ribonucleoprotein (RNP) complex composed of the Cas9 endonuclease, a CRISPR-derived RNA (crRNA), and a trans-activating CRISPR RNA (tracrRNA). Around 100 anti-CRISPR proteins that inhibit CRISPR-Cas systems have been identified, and the mechanisms by which they act are increasingly being elucidated. However, the inhibitory mechanisms of many Acrs, including AcrIIA7, remain poorly understood. Here, we present the structure of AcrIIA7 and uncover a previously unrecognized mechanism by which it inhibits Cas9 function. Structural and biochemical analyses reveal that AcrIIA7 specifically binds to tracrRNA, preventing its association with crRNA and thereby blocking formation of the active Cas9 RNP complex. This tracrRNA hijacking mechanism represents a unique strategy for CRISPR inhibition, in which an anti-CRISPR protein targets an RNA scaffold essential for Cas9 activation rather than interacting directly with the Cas9 protein. Our findings provide the first structural insight into tracrRNA-targeted anti-CRISPR activity and highlight RNA-RNA interaction interfaces as vulnerable nodes in CRISPR-Cas immunity.