Tang Honghai, Yuan Hui, Du Wenhao, Li Gan, Xue Dongmei, Huang Qiang
State Key Laboratory of Genetic Engineering, Shanghai Engineering Research Center of Industrial Microorganisms, Ministry of Education Engineering Research Centre of Gene Technology, School of Life Sciences, Fudan University, Shanghai, China.
Multiscale Research Institute of Complex Systems, Fudan University, Shanghai, China.
Front Mol Biosci. 2021 Apr 21;8:653262. doi: 10.3389/fmolb.2021.653262. eCollection 2021.
CRISPR-Cas9 is a powerful tool for target genome editing in living cells. Significant advances have been made to understand how this system cleaves target DNA. HNH is a nuclease domain, which shares structural similarity with the HNH endonuclease characterzied by a beta-beta-alpha-metal fold. Therefore, based on one- and two-metal-ion mechanisms, homology modeling and molecular dynamics (MD) simulation are suitable tools for building an atomic model of Cas9 in the DNA cleavage state. Here, by modeling and MD, we presented an atomic model of SpCas9-sgRNA-DNA complex with the cleavage state. This model shows that the HNH and RuvC conformations resemble their DNA cleavage state where the active-sites in the complex coordinate with DNA, Mg ions, and water. Among them, residues D10, E762, H983, and D986 locate at the first shell of the RuvC active-site and interact with the ions directly, residues H982 or/and H985 are general (Lewis) bases, and the coordinated water is located at the positions for nucleophilic attack of the scissile phosphate. Meanwhile, this catalytic model led us to engineer a new SpCas9 variant (SpCas9-H982A + H983D) with reduced off-target effects. Thus, our study provided new mechanistic insights into the CRISPR-Cas9 system in the DNA cleavage state and offered useful guidance for engineering new CRISPR-Cas9 editing systems with improved specificity.
CRISPR-Cas9是一种用于活细胞中靶向基因组编辑的强大工具。在理解该系统如何切割靶DNA方面已经取得了重大进展。HNH是一种核酸酶结构域，与具有β-β-α-金属折叠特征的HNH内切核酸酶具有结构相似性。因此，基于单金属离子和双金属离子机制，同源建模和分子动力学（MD）模拟是构建处于DNA切割状态的Cas9原子模型的合适工具。在此，通过建模和MD，我们展示了处于切割状态的SpCas9-sgRNA-DNA复合物的原子模型。该模型表明，HNH和RuvC的构象类似于它们的DNA切割状态，其中复合物中的活性位点与DNA、镁离子和水配位。其中，D10、E762、H983和D986残基位于RuvC活性位点的第一配位层并直接与离子相互作用，H982或/和H985残基是通用（路易斯）碱，配位水位于可切割磷酸酯亲核攻击的位置。同时，这种催化模型使我们设计出一种具有降低脱靶效应的新型SpCas9变体（SpCas9-H982A + H983D）。因此，我们的研究为处于DNA切割状态的CRISPR-Cas9系统提供了新的机制见解，并为设计具有更高特异性的新型CRISPR-Cas9编辑系统提供了有用的指导。
