The anti-CRISPR protein AcrIE8.1 inhibits the type I-E CRISPR-Cas system by directly binding to the Cascade subunit...

CRISPR-Cas systems provide adaptive immunity to bacteria by recognizing and destroying foreign genetic elements. The type I-E CRISPR-Cas system utilizes a multi-subunit Cascade complex to detect target DNA and recruit the Cas3 nuclease for degradation. To overcome this defense, bacteriophages have evolved anti-CRISPR (Acr) proteins that inhibit various steps of the CRISPR interference pathway. Here, we determined the crystal structure of AcrIE8.1, an uncharacterized Acr, revealing it binds to Cas11, a Cascade subunit, to disrupt function. AcrIE8.1 has a compact fold with a defined Cas11-binding interface, suggesting a unique inhibitory mechanism among AcrIE proteins. These findings highlight Cas11 as a critical target for Acr-mediated immune evasion. Impact statement Through a combination of structural and biochemical analyses, we demonstrate that AcrIE8.1 directly binds to the Cas11 subunit of the Cascade complex to inhibit the CRISPR-Cas system. This represents a novel inhibitory strategy not previously observed among AcrIE proteins.