A dual gating mechanism controls target‐strand cleavage in Cas12j: Implications for engineering efficient nickases

The rapid expansion of CRISPR technologies has unveiled a diverse repertoire of RNA-guided endonucleases, among them the compact Cas12j, which has emerged as a promising genome-editing tool. Cas12j cleaves the two DNA strands sequentially; however, the molecular mechanism that regulates this cleavage remains incompletely understood. Here, we combine extensive all-atom molecular dynamics simulations with well-tempered metadynamics, totaling approximately 175 μs of cumulative sampling, to investigate how target-strand accessibility to the catalytic site is controlled. Our results are in agreement with previous experimental observations, and furthermore reveal new mechanistic details that are difficult to access experimentally, namely a coordinated dual-barrier mechanism governing target-strand accessibility that can be fine-tuned through targeted mutations in the α7-helix and/or the REC2 loop. These findings provide a mechanistic basis for tuning Cas12j activity along the nuclease-to-nickase spectrum, supporting the rational engineering of genome-editing tools with controlled target-strand cleavage kinetics, and offering a path toward applications that bypass dependence on Non-Homologous End Joining (NHEJ) or Homology-Directed Repair (HDR).