Structural Molecular Biology Group, Novo Nordisk Foundation Centre for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen, Denmark.
Department of Chemistry & Nanoscience Centre, University of Copenhagen, Universitetsparken 5, 2100 Copenhagen, Denmark.
Cell. 2018 Dec 13;175(7):1856-1871.e21. doi: 10.1016/j.cell.2018.10.045. Epub 2018 Nov 29.
Cas12a, also known as Cpf1, is a type V-A CRISPR-Cas RNA-guided endonuclease that is used for genome editing based on its ability to generate specific dsDNA breaks. Here, we show cryo-EM structures of intermediates of the cleavage reaction, thus visualizing three protein regions that sense the crRNA-DNA hybrid assembly triggering the catalytic activation of Cas12a. Single-molecule FRET provides the thermodynamics and kinetics of the conformational activation leading to phosphodiester bond hydrolysis. These findings illustrate why Cas12a cuts its target DNA and unleashes unspecific cleavage activity, degrading ssDNA molecules after activation. In addition, we show that other crRNAs are able to displace the R-loop inside the protein after target DNA cleavage, terminating indiscriminate ssDNA degradation. We propose a model whereby the conformational activation of the enzyme results in indiscriminate ssDNA cleavage. The displacement of the R-loop by a new crRNA molecule will reset Cas12a specificity, targeting new DNAs.
Cas12a，也被称为 Cpf1，是一种 V-A 型 CRISPR-Cas RNA 指导的内切核酸酶，由于其能够产生特定的 dsDNA 断裂，因此被用于基因组编辑。在这里，我们展示了切割反应中间产物的低温电镜结构，从而可视化了三个蛋白区域，这些区域能够感知 crRNA-DNA 杂交组装，从而触发 Cas12a 的催化激活。单分子 FRET 提供了导致磷酸二酯键水解的构象激活的热力学和动力学。这些发现说明了为什么 Cas12a 会切割其靶 DNA 并释放非特异性切割活性，在激活后降解 ssDNA 分子。此外，我们还表明，其他 crRNA 能够在靶 DNA 切割后置换蛋白质内的 R 环，终止无差别 ssDNA 降解。我们提出了一个模型，其中酶的构象激活导致无差别 ssDNA 切割。新的 crRNA 分子置换 R 环将重置 Cas12a 的特异性，靶向新的 DNA。
