Abstract
Cas12a (Cpf1) is a bacterial RNA-guided nuclease used widely for genome editing and diagnostic applications. In bacteria, Cas12a enzymes can be inhibited by bacteriophage-derived proteins, anti-CRISPRs (Acrs), to thwart clustered regularly interspaced short palindromic repeat (CRISPR) adaptive immune systems. How these inhibitors disable Cas12a by preventing programmed DNA cleavage is unknown. We show that three inhibitors (AcrVA1, AcrVA4 and AcrVA5) block Cas12a activity using functionally distinct mechanisms, including a previously unobserved enzymatic strategy. AcrVA4 and AcrVA5 inhibit double-stranded DNA (dsDNA) recognition with AcrVA4 driving Cas12a dimerization. In contrast, AcrVA1 is a multiple-turnover inhibitor that triggers cleavage of the target recognition sequence of the Cas12a-bound guide RNA to irreversibly inactivate the Cas12a complex. These distinct mechanisms equip bacteriophage with tools to evade CRISPR-Cas12a and support biotechnological applications where multiple-turnover enzymatic inhibition of Cas12a are desirable.
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