Conformational Dynamics and Cleavage Sites of Cas12a Are Modulated by Complementarity between crRNA and DNA
Conformational Dynamics and Cleavage Sites of Cas12a Are Modulated by Complementarity between crRNA and DNA
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Cas12a 的构象动力学和切割位点受 crRNA 和 DNA 之间的互补性调节。
DOI：
10.1016/j.isci.2019.08.005
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发表时间：
2019-09-27
期刊：
影响因子：
5.8
通讯作者：
Chen, Chunlai
中科院分区：
文献类型：
--
作者：
Zhang, Lujia;Sun, Ruirui;Chen, Chunlai
Cas 12 a是一种RNA引导的核酸内切酶，在基因组编辑和工程中显示出巨大的潜力和优于Cas9的优势。在这里，我们建立了一个定量动力学方案来描述Cas 12 a/crRNA/dsDNA三元复合物的构象动力学。Cas 12 a复合物的高度动态性质，包括它们的可逆形成、分解和在不同构象状态之间的转变，可能是促成其高特异性的关键方面之一。当Cas 12 a的DNA酶活化后，非靶链的切割位点从DNA双链体释放时，非靶链被切割。切割的非靶链稳定靶链切割前状态，以允许随后的切割并确保两条DNA链以明确定义的顺序切割。crRNA和DNA之间的互补性程度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Cas 12 a的工作机制并优化其用于基因组工程提供了见解。
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.