Mechanistic Insights into the Cis- and Trans-acting Deoxyribonuclease Activities of Cas12a

CRISPR-Cas12a (Cpf1) is an RNA-guided DNA-cutting nuclease that has been repurposed for genome editing. Upon target DNA binding, Cas12a cleaves both the target DNA in cis and non-target single stranded DNAs (ssDNA) in trans. To elucidate the molecular basis for both deoxyribonuclease cleavage modes, we performed structural and biochemical studies on Francisella novicida Cas12a. We show that crRNA-target DNA hybridization conformationally activates Cas12a, triggering its trans-acting, non-specific, single-stranded deoxyribonuclease activity. In turn, cis-cleavage of double-stranded DNA targets is a result of PAM-dependent DNA duplex unwinding and ordered sequential cleavage of the non-target and target DNA strands. Cas12a releases the PAM-distal DNA cleavage product and remains bound to the PAM-proximal DNA cleavage product in a catalytically competent, trans-active state. Together, these results provide a revised model for the molecular mechanism of both the cis- and trans-acting deoxyribonuclease activities of Cas12a enzymes, allowing their further exploitation as genome editing tools.