The Conformational Dynamics of Cas9 Governing DNA Cleavage Are Revealed by Single-Molecule FRET
The Conformational Dynamics of Cas9 Governing DNA Cleavage Are Revealed by Single-Molecule FRET
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单分子 FRET 揭示了 Cas9 控制 DNA 切割的构象动力学。
DOI：
10.1016/j.celrep.2017.12.048
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发表时间：
2018-01-09
期刊：
影响因子：
8.8
通讯作者：
Chen, Chunlai
中科院分区：
文献类型：
--
作者：
Yang, Mengyi;Peng, Sijia;Chen, Chunlai
Cas9的脱靶结合和裂解是其应用的主要挑战。Cas9的构象动力学如何在靶向和脱靶条件下控制其核酸酶活性仍然很大程度上未知。通过分子内单分子荧光共振能量转移测量，我们发现Cas9在apo、sgrna结合和dsDNA/ sgrna结合形式中自发地在三种主要构象状态之间转移，主要反映了催化HNH结构域的显著构象迁移性。我们还发现了HNH结构域和pam远端RNA/DNA异双工之间令人惊讶的长距离变构通信，以确保催化位点的正确定位，这表明一种独特的校对机制是DNA切割前的最后一个检查点。几个Cas9残基可能介导变构通信和校对步骤。通过在pam -远端引入突变来调节Cas9和异双工之间的相互作用，为改进和优化CRISPR/Cas9工具箱提供了另一种途径。
Off-target binding and cleavage by Cas9 pose major challenges in its application. How the conformational dynamics of Cas9 govern its nuclease activity under on-and off-target conditions remains largely unknown. Here, using intra-molecular single-molecule fluorescence resonance energy transfer measurements, we revealed that Cas9 in apo, sgRNA-bound, and dsDNA/sgRNA-bound forms spontaneously transits among three major conformational states, mainly reflecting significant conformational mobility of the catalytic HNH domain. We also uncovered surprising long-range allosteric communication between the HNH domain and the RNA/DNA hetero-duplex at the PAM-distal end to ensure correct positioning of the catalytic site, which demonstrated that a unique proofreading mechanism served as the last checkpoint before DNA cleavage. Several Cas9 residues were likely to mediate the allosteric communication and proofreading step. Modulating interactions between Cas9 and heteroduplex at the PAM-distal end by introducing mutations on these sites provides an alternative route to improve and optimize the CRISPR/Cas9 toolbox.