CRISPR-Cas9 Mediated DNA Unwinding Detected Using Site-Directed Spin Labeling.
CRISPR-Cas9 Mediated DNA Unwinding Detected Using Site-Directed Spin Labeling.
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使用定点自旋标记检测 CRISPR-Cas9 介导的 DNA 解旋。
DOI：
10.1021/acschembio.6b01137
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发表时间：
2017
影响因子：
4
通讯作者：
Qin,PeterZ
中科院分区：
文献类型：
--
作者：
Tangprasertchai,NarinS;DiFelice,Rosa;Zhang,Xiaojun;Slaymaker,IanM;VazquezReyes,Carolina;Jiang,Wei;Rohs,Remo;Qin,PeterZ
RNA引导的CRISPR-Cas9核酸酶已经彻底改变了基因组工程，但其DNA靶标选择的机制尚未完全了解。Cas9靶标识别的关键步骤涉及DNA双链体的解旋以形成三链R环结构。本文报道的工作证明了通过定点自旋标记和分子动力学模拟的组合直接检测Cas9介导的DNA解旋。结果支持这样一种模型，其中解绕的非靶链通过位于Cas9的两个核酸酶结构域之间的带正电荷的补丁而稳定，并且揭示了在DNA主链断裂时柔性沿着解绕的非靶链的不均匀增加。这项工作建立了自旋标记和分子动力学的协同组合，以直接监测Cas9介导的DNA构象变化，并在Cas9功能的不同阶段产生关于靶DNA的信息，从而推进了对CRISPR-Cas9的机制理解，并有助于未来的技术发展。
The RNA-guided CRISPR–Cas9 nuclease has revolutionized genome engineering, yet its mechanism for DNA target selection is not fully understood. A crucial step in Cas9 target recognition involves unwinding of the DNA duplex to form a three-stranded R-loop structure. Work reported here demonstrates direct detection of Cas9-mediated DNA unwinding by a combination of site-directed spin labeling and molecular dynamics simulations. The results support a model in which the unwound nontarget strand is stabilized by a positively charged patch located between the two nuclease domains of Cas9 and reveal uneven increases in flexibility along the unwound nontarget strand upon scissions of the DNA backbone. This work establishes the synergistic combination of spin-labeling and molecular dynamics to directly monitor Cas9-mediated DNA conformational changes and yields information on the target DNA in different stages of Cas9 function, thus advancing mechanistic understanding of CRISPR–Cas9 and aiding future technological development.