Department of Pharmaceutical Sciences, UNT System College of Pharmacy, University of North Texas Health Science Center, Fort Worth, United States.
College of Chemistry and Chemical Engineering, Shanghai University of Engineering Science, Shanghai, China.
Elife. 2019 Jul 30;8:e46500. doi: 10.7554/eLife.46500.
The CRISPR-associated endonuclease Cas9 from (SpyCas9), along with a programmable single-guide RNA (sgRNA), has been exploited as a significant genome-editing tool. Despite the recent advances in determining the SpyCas9 structures and DNA cleavage mechanism, the cleavage-competent conformation of the catalytic HNH nuclease domain of SpyCas9 remains largely elusive and debatable. By integrating computational and experimental approaches, we unveiled and validated the activated Cas9-sgRNA-DNA ternary complex in which the HNH domain is neatly poised for cleaving the target DNA strand. In this catalysis model, the HNH employs the catalytic triad of D839-H840-N863 for cleavage catalysis, rather than previously implicated D839-H840-D861, D837-D839-H840, or D839-H840-D861-N863. Our study contributes critical information to defining the catalytic conformation of the HNH domain and advances the knowledge about the conformational activation underlying Cas9-mediated DNA cleavage.
来自 （SpyCas9）的 CRISPR 相关内切酶 Cas9 与可编程的单指导 RNA（sgRNA）一起，已被开发为一种重要的基因组编辑工具。尽管最近在确定 SpyCas9 结构和 DNA 切割机制方面取得了进展，但 SpyCas9 的催化 HNH 核酸酶结构域的切割活性构象在很大程度上仍然难以捉摸和有争议。通过整合计算和实验方法，我们揭示并验证了激活的 Cas9-sgRNA-DNA 三元复合物，其中 HNH 结构域整齐地准备好切割靶 DNA 链。在这个催化模型中，HNH 采用催化三联体 D839-H840-N863 进行催化，而不是先前涉及的 D839-H840-D861、D837-D839-H840 或 D839-H840-D861-N863。我们的研究为定义 HNH 结构域的催化构象提供了关键信息，并推进了 Cas9 介导的 DNA 切割的构象激活的知识。
