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Cas12j cleaves the two DNA strands sequentially; however, the molecular mechanism that regulates this cleavage remains incompletely understood. Here, we combine extensive all\u2010atom molecular dynamics simulations with well\u2010tempered metadynamics, totaling approximately 175\u2009\u03bcs of cumulative sampling, to investigate how target\u2010strand 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\u2010barrier mechanism governing target\u2010strand accessibility that can be fine\u2010tuned through targeted mutations in the \u03b17\u2010helix and\/or the REC2 loop. These findings provide a mechanistic basis for tuning Cas12j activity along the nuclease\u2010to\u2010nickase spectrum, supporting the rational engineering of genome\u2010editing tools with controlled target\u2010strand cleavage kinetics, and offering a path toward applications that bypass dependence on Non\u2010Homologous End Joining (NHEJ) or Homology\u2010Directed Repair (HDR).<\/jats:p>","DOI":"10.1002\/pro.70792","type":"journal-article","created":{"date-parts":[[2026,9,15]],"date-time":"2026-09-15T13:52:42Z","timestamp":1789480362000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["A dual gating mechanism controls target\u2010strand cleavage in Cas12j: Implications for engineering efficient nickases"],"prefix":"10.1002","volume":"35","author":[{"ORCID":"https:\/\/orcid.org\/0009-0004-7686-614X","authenticated-orcid":false,"given":"Ilias 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