Mechanistic Decoupling of the cis - and trans -Cleavage Activities of Cas12a via Phosphorothioate-Induced Kinetic Trapping

Abstract

CRISPR-Cas12a is widely utilized for genome engineering and nucleic acid diagnostics, being distinguished by its indiscriminate single-stranded DNA (ssDNA) trans-cleavage activity triggered by its specific cis-target recognition. However, the precise kinetic coordination between these dual catalytic modes remains unclear because of methodological limitations, which preclude simultaneous monitoring of both activities. Here, we established a real-time, dual-wavelength fluorescence reporter system to dissect these dynamics utilizing phosphorothioate (PS) backbone modifications as chemical probes to interrogate enzyme turnover. We identified a functional decoupling and an asymmetric competitive mechanism strictly governed by “channel occupancy”. Specifically, we found that the PS modification of the cis-target abolished the trans-activity via a “product release gating” mechanism, where high-affinity product retention occluded the active site. Furthermore, we identified a critical length-dependent regulatory regime for ssDNA reporters, while short, noncleavable ligands (5-nt) acted as passive spectator molecules, and long analogues (30-nt) functioned as potent competitive inhibitors. The long ligands induced an irreversible “kinetic trap”, creating a nonproductive complex where the enzyme was permanently sequestered because of the lack of cleavage-mediated release. These findings demonstrate that the availability of the RuvC catalytic channel is determined not by induced fit binding but by the chemical cleavability of the occupant. This study establishes an integrated Cas12a-regulated kinetic model and systematically investigates the simultaneous effects of PS-modification on the cis- and trans-hydrolytic activities of Cas12a. The findings provide a theoretical framework and additional insights for developing more precise gene-editing tools and designing Cas12a-based in vitro diagnostic platforms.