Real-time observation of flexible domain movements in Cas9
Real-time observation of flexible domain movements in Cas9
复制标题
DOI：
10.1101/122069
复制
发表时间：
2017-03
期刊：
影响因子：
--
通讯作者：
Saki Osuka;Kazushi Isomura;Shohei Kajimoto;Tomotaka Komori;H. Nishimasu;T. Shima;O. Nureki;S. Uemura
中科院分区：
文献类型：
--
作者：
Saki Osuka;Kazushi Isomura;Shohei Kajimoto;Tomotaka Komori;H. Nishimasu;T. Shima;O. Nureki;S. Uemura
CRISPR相关蛋白Cas9是一种广泛使用的基因组编辑工具，它通过单引导RNA(SgRNA)的辅助识别和切割目标DNA。结构研究证明了Cas9的多结构域结构，以及与sgRNA和靶DNA结合时的顺序结构域移动。这些研究也暗示了域之间的灵活性，但这些灵活的运动是否发生在解决方案中尚不清楚。在这里，我们使用单分子FRET直接观察到多个Cas9结构域的动态波动。灵活的结构域移动使Cas9能够采用晶体结构中捕获的以外的瞬时构象。重要的是，Cas9中的HNH核酸酶结构域只有在这种灵活的运动中才进入DNA裂解位置，这表明这种灵活性在DNA裂解过程中的重要性。我们的FRET数据还揭示了apo-Cas9的构象灵活性，这可能在与sgRNA的组装中发挥作用。总体而言，我们的结果强调了结构域波动在驱动Cas9催化的DNA切割中的潜在作用。
The CRISPR-associated protein Cas9 is a widely used genome editing tool that recognizes and cleaves target DNA through the assistance of a single-guide RNA (sgRNA). Structural studies have demonstrated the multi-domain architecture of Cas9 and sequential domain movements upon binding to the sgRNA and the target DNA. These studies also hinted at the flexibility between domains, but whether these flexible movements occur in solution is unclear. Here, we directly observed dynamic fluctuations of multiple Cas9 domains, using single-molecule FRET. The flexible domain movements allow Cas9 to adopt transient conformations beyond those captured in the crystal structures. Importantly, the HNH nuclease domain in Cas9 only accessed the DNA cleavage position during such flexible movements, suggesting the importance of this flexibility in the DNA cleavage process. Our FRET data also revealed the conformational flexibility of apo-Cas9, which may play a role in the assembly with the sgRNA. Collectively, our results highlight the potential role of domain fluctuations in driving Cas9-catalyzed DNA cleavage.