A conformational checkpoint between DNA binding and cleavage by CRISPR-Cas9.
A conformational checkpoint between DNA binding and cleavage by CRISPR-Cas9.
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DOI：
10.1126/sciadv.aao0027
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发表时间：
2017-08
期刊：
影响因子：
13.6
通讯作者：
Yildiz A
中科院分区：
文献类型：
--
作者：
Dagdas YS;Chen JS;Sternberg SH;Doudna JA;Yildiz A
Cas9的构象检查点使得能够进行中靶切割，同时阻断在脱靶位点处进入活性状态。Cas9核酸内切酶通过编程其单向导RNA而广泛用于基因组工程应用，并且正在进行的工作旨在提高DNA靶向的准确性和效率。Cas9的DNA切割由HNH核酸酶结构域的构象状态控制，但是控制在靶DNA处的HNH活化同时降低在脱靶位点处的切割活性的机制仍然知之甚少。使用单分子Förster共振能量转移，我们确定了化脓链球菌Cas9的中间状态，代表DNA结合和切割之间的构象检查点。在DNA结合时，HNH结构域在对接到其活性状态之前在多种构象之间转变。HNH对接需要二价阳离子，但不需要链断裂，并且这种对接构象在DNA切割后持续存在。DNA靶和指导RNA之间的序列错配防止从检查点中间体转变为活性构象，从而提供在稳定结合的脱靶位点处选择性避免DNA切割。
A conformational checkpoint of Cas9 enables on-target cleavage while blocking access to the active state at off-target sites. The Cas9 endonuclease is widely used for genome engineering applications by programming its single-guide RNA, and ongoing work is aimed at improving the accuracy and efficiency of DNA targeting. DNA cleavage of Cas9 is controlled by the conformational state of the HNH nuclease domain, but the mechanism that governs HNH activation at on-target DNA while reducing cleavage activity at off-target sites remains poorly understood. Using single-molecule Förster resonance energy transfer, we identified an intermediate state of Streptococcus pyogenes Cas9, representing a conformational checkpoint between DNA binding and cleavage. Upon DNA binding, the HNH domain transitions between multiple conformations before docking into its active state. HNH docking requires divalent cations, but not strand scission, and this docked conformation persists following DNA cleavage. Sequence mismatches between the DNA target and guide RNA prevent transitions from the checkpoint intermediate to the active conformation, providing selective avoidance of DNA cleavage at stably bound off-target sites.
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