Cas12a is an RNA-guided endonuclease, which displays great potentials and several advantages over the well-known Cas9 in genome editing and engineering. Here, we established a quantitative kinetic scheme to describe the conformational dynamics of Cas12a/crRNA/dsDNA ternary complexes. The highly dynamic nature of Cas12a complexes, including their reversible formation, disassembly, and transition between different conformational states, is likely to be one of the key aspects contributing to their high specificity. The non-target strand is cleaved when its cleavage sites are released from DNA duplex after DNase activation of Cas12a. Cleaved non-target strand stabilizes target strand pre-cleavage states to permit subsequent cleavage and to ensure two DNA strands cleaved in a well-defined order. The extent of complementarity between crRNA and DNA modulates the relative stabilities of target strand pre-cleavage states targeting different cleavage sites. Our discoveries provide insights to fully elucidate the working mechanisms of Cas12a and to optimize it for genome engineering.

Cas12a是一种RNA引导的核酸内切酶，在基因组编辑和工程设计方面，与知名的Cas9相比，它具有巨大的潜力和多项优势。在这里，我们建立了定量动力学方案来描述Cas12a / crRNA / dsDNA三元复合物的构象动力学。Cas12a复合物的高度动态性质，包括其可逆的形成，分解和不同构象状态之间的过渡，很可能是促成其高特异性的关键方面之一。当DNA酶激活Cas12a后，非切割链的切割位点从DNA双链体中释放出来时，非切割链被切割。切割的非目标链可稳定目标链的预切割状态，以允许后续切割并确保两条DNA链以明确的顺序切割。crRNA和DNA之间的互补程度可调节靶向不同切割位点的靶链预切割状态的相对稳定性。我们的发现为全面阐明Cas12a的工作机制并针对基因组工程进行优化提供了见识。