Key Role of an Alpha-helical Lid in Driving the Target DNA toward Catalysis in CRISPR-Cas12a

CRISPR-Cas12a is an emerging gene editing system that came to the forefront with innovative applications such as rapid and ultrasensitive nucleic acid detection. At the core of this molecular complex, the RNA-guided Cas12a enzyme cleaves double-stranded DNA using a single catalytic cleft in the RuvC domain. This opens an overarching question on how the spatially distant DNA target strand (TS) traverses toward the RuvC domain for its accommodation and subsequent cleavage in the catalytic core. Here, continuous tens of microsecond-long molecular dynamics and free-energy simulations reveal that an α-helical lid, located within the RuvC domain, plays a pivotal role in the traversal of the TS by anchoring the crRNA:TS hybrid and elegantly guiding the TS toward the RuvC core, as also corroborated by DNA cleavage experiments. In this mechanism, the Rec2 domain drives the DNA target strand toward the RuvC catalytic cleft, owing to concerted motions with the Nuc domain. While the REC2 domain pushes the TS inward into the core of the complex with its short alpha helices, the Nuc domain aids the bending and accommodation of the TS within the RuvC core by bending inward. The identified intermediates provide information on the critical residues involved in the biophysical process, holding promises for future engineering strategies aimed at improving the overall Cas12a activity.