Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo, Japan.
Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo, Japan
EMBO J. 2018 May 15;37(10). doi: 10.15252/embj.201796941. Epub 2018 Apr 12.
The CRISPR-associated protein Cas9 is widely used for genome editing because it cleaves target DNA through the assistance of a single-guide RNA (sgRNA). Structural studies have revealed the multi-domain architecture of Cas9 and suggested sequential domain movements of Cas9 upon binding to the sgRNA and the target DNA These studies also hinted at the flexibility between domains; however, it remains unclear whether these flexible movements occur in solution. Here, we directly observed dynamic fluctuations of multiple Cas9 domains, using single-molecule FRET We found that the flexible domain movements allow Cas9 to adopt transient conformations beyond those captured in the crystal structures. Importantly, the HNH nuclease domain 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.
CRISPR 相关蛋白 Cas9 被广泛用于基因组编辑，因为它可以在单指导 RNA（sgRNA）的辅助下切割靶 DNA。结构研究揭示了 Cas9 的多结构域架构，并提出了 Cas9 在与 sgRNA 和靶 DNA 结合时的顺序域运动。这些研究还暗示了域之间的灵活性；然而，目前尚不清楚这些灵活的运动是否发生在溶液中。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。我们发现，灵活的结构域运动使 Cas9 能够采用超越晶体结构中捕获的瞬态构象。重要的是，只有在这种灵活的运动过程中，HNH 核酸酶结构域才能进入 DNA 切割位置，这表明这种灵活性在 DNA 切割过程中的重要性。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。总之，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。
