Direct observation of the formation of a CRISPR-Cas12a R-loop complex at the single-molecule level
Direct observation of the formation of a CRISPR-Cas12a R-loop complex at the single-molecule level
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在单分子水平上直接观察 CRISPR-Cas12a R 环复合物的形成
DOI：
10.1039/c9cc08325a
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发表时间：
2020-02-18
影响因子：
4.9
通讯作者：
Lou, Jizhong
中科院分区：
文献类型：
--
作者：
Cui, Yang;Tang, Yangchao;Lou, Jizhong
在这里，我们开发了一种基于光镊的单分子操作检测方法来检测Cas12a系统中R环复合物的形成并表征其热力学稳定性。我们发现，R环复合物的形成诱导了含有靶序列的DNA发夹的两步解折叠，非靶序列与Cas12a松散地结合并且可以容易地从复合物中释放，并且Cas12a的Nuc结构域在靶结合和R环形成中起关键作用。
Here, we develop an optical tweezers-based single-molecule manipulation assay to detect the formation of an R-loop complex in the Cas12a system and characterize its thermodynamic stability. We found that the formation of the R-loop complex induces a two-step unfolding of a DNA hairpin containing the target sequence, the non-target sequence binds loosely to Cas12a and can be easily released from the complex, and the Nuc domain of Cas12a plays key roles in target binding and R-loop formation.