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

HIGHLIGHTSO_LITarget ssDNA binding allosterically induces unblocking of the RuvC active site\nC_LIO_LIPAM binding facilitates unwinding of dsDNA targets\nC_LIO_LINon-target DNA strand cleavage is prerequisite for target DNA strand cleavage\nC_LIO_LIAfter DNA cleavage, Cas12a releases the PAM-distal DNA product\nC_LI\n\nSUMMARYCRISPR-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 how crRNA-target DNA strand 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 Cas12a enzymes that explains their cis- and trans-acting deoxyribonuclease activities, and additionally contribute to improving Cas12a-based genome editing.