Direct observation of DNA target searching and cleavage by CRISPR-Cas12a.
Direct observation of DNA target searching and cleavage by CRISPR-Cas12a.
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DOI：
10.1038/s41467-018-05245-x
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发表时间：
2018-07-17
影响因子：
16.6
通讯作者：
Bae S
中科院分区：
文献类型：
--
作者：
Jeon Y;Choi YH;Jang Y;Yu J;Goo J;Lee G;Jeong YK;Lee SH;Kim IS;Kim JS;Jeong C;Lee S;Bae S
Cas12a（也称为Cpf1）是V-A型CRISPR效应rna引导的DNA内切酶的代表，它为基因组编辑提供了一种替代II型CRISPR - cas9的方法。先前的研究表明，Cas12a具有不同于Cas9的独特特征，但Cas12a寻找靶标和切割DNA的详细机制尚不清楚。在这里，我们采用单分子荧光法直接观察了这整个过程，研究了来自酸胺球菌（Acidaminococcus sp.）的Cas12a。我们确定AsCas12a核糖核蛋白通过沿着长链DNA分子的一维扩散来寻找它们的靶位点，并以明确的顺序诱导两条DNA链的切割，从非靶链开始。此外，与Cas9 PAM相比，AsCas12a的原间隔邻近基序（protospacer-邻基序，PAM）在r环形成过程中对DNA解绕的贡献有限，在DNA切割过程中的作用可以忽略不计。Cas12a是一种rna引导的DNA内切酶，其靶标搜索和DNA切割的详细机制尚不清楚。在这里，作者使用单分子荧光分析来显示Cas12a搜索它们的靶位点。
Cas12a (also called Cpf1) is a representative type V-A CRISPR effector RNA-guided DNA endonuclease, which provides an alternative to type II CRISPR–Cas9 for genome editing. Previous studies have revealed that Cas12a has unique features distinct from Cas9, but the detailed mechanisms of target searching and DNA cleavage by Cas12a are still unclear. Here, we directly observe this entire process by using single-molecule fluorescence assays to study Cas12a from Acidaminococcus sp. (AsCas12a). We determine that AsCas12a ribonucleoproteins search for their on-target site by a one-dimensional diffusion along elongated DNA molecules and induce cleavage in the two DNA strands in a well-defined order, beginning with the non-target strand. Furthermore, the protospacer-adjacent motif (PAM) for AsCas12a makes only a limited contribution of DNA unwinding during R-loop formation and shows a negligible role in the process of DNA cleavage, in contrast to the Cas9 PAM. Cas12a is a RNA-guided DNA endonuclease whose detailed mechanisms of target searching and DNA cleavage remained unclear. Here authors use single-molecule fluorescence assays to show that Cas12a searches for their on-target site.