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

Abstract

Abstract 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).