CRISPR-Cas12a, an RNA-guided DNA targeting endonuclease, has been widely used for genome editing and nucleic acid detection. As part of the essential processes for both of these applications, the two strands of double-stranded DNA are sequentially cleaved by a single catalytic site of Cas12a, but the mechanistic details that govern the generation of complete breaks in double-stranded DNA remain to be elucidated. Here, using single-molecule fluorescence resonance energy transfer assay, we identified two conformational intermediates that form consecutively following the initial cleavage of the nontarget strand. Specifically, these two intermediates are the result of further unwinding of the target DNA in the protospacer-adjacent motif (PAM)–distal region and the subsequent binding of the target strand to the catalytic site. Notably, the PAM-distal DNA unwound conformation was stabilized by Mg²⁺ ions, thereby significantly promoting the binding and cleavage of the target strand. These findings enabled us to propose a Mg²⁺-dependent kinetic model for the mechanism whereby Cas12a achieves cleavage of the target DNA, highlighting the presence of conformational rearrangements for the complete cleavage of the double-stranded DNA target.

CRISPR-Cas12a是一种RNA引导的DNA靶向核酸内切酶，已广泛用于基因组编辑和核酸检测。作为这两种应用的基本过程的一部分，双链 DNA 的两条链被 Cas12a 的单个催化位点顺序切割，但控制双链 DNA 完全断裂产生的机制细节仍有待研究。阐明了。在这里，使用单分子荧光共振能量转移测定，我们鉴定了在非目标链初始切割后连续形成的两种构象中间体。具体来说，这两个中间体是原型间隔子相邻基序 (PAM) 远端区域中靶 DNA 进一步解旋以及随后靶链与催化位点结合的结果。值得注意的是，PAM-远端DNA解绕构象被Mg ²⁺离子稳定，从而显着促进靶链的结合和切割。这些发现使我们能够针对 Cas12a 实现靶标 DNA 切割的机制提出 Mg ²⁺依赖性动力学模型，强调了双链 DNA 靶标完全切割时构象重排的存在。