Cas9-sgRNA复合物靶向切割DNA的分子基础

Recently, CRISPR technology based on the Cas9-sgRNA complex has become a revolutionary tool for genome editing. However, problems such as off-target effects and low-transmission efficiency have seriously hampered its applications. Therefore, it is urgent to clarify the molecular basis for the targeting cleavage of DNA by the Cas9-sgRNA complex, in order to provide scientific guidelines for optimization and improvement of the Cas9-sgRNA system. To the end, in this proposal we plan to firstly use cryo-electron microscopy (cryo-EM) to determine the near-atomic-resolution structure of the Cas9-sgRNA-DNA ternary complex with complete target DNA strands, and thereby to construct atomic models that describe the DNA target binding and cleavage reaction by Cas9-sgRNA. Next, we will perform structural modeling to analyze the molecular driving forces of the target binding and cleavage reaction, and then to establish quantitative models for identifying the key molecular groups in these two processes. Finally, guided by the modeling results, we will design miniaturized Cas9 mutants by computationally removing unnecessary amino acids. The study of this project is expected to develop a new theory that clarifies the molecular basis of the target binding and cleavage reaction by Cas9-sgRNA, to reveal the key molecular groups in these two processes, and eventually to develop miniaturized Cas9 mutants with high transmission-efficiency and specificity. So, this project will provide new scientific insights and computational tools for the optimization and improvement of the Cas9-sgRNA system, and thus promote the active applications of the CRISPR technology in biomedicine.

近年，基于Cas9-sgRNA复合物的CRISPR技术已成为革命性的基因组编辑工具。但是，脱靶效应和低输送效率等问题正严重阻碍该技术的应用。因此，迫切需要研究清楚该复合物靶向切割DNA的分子基础，为系统的优化改造提供科学依据。为解决此问题，本项目拟先用冷冻电镜解析含完整DNA双链的Cas9-sgRNA-DNA三元复合物的近原子分辨率结构，构建描述DNA靶标结合与剪切反应过程的原子模型；接着，用结构建模方法分析靶标结合与剪切反应的分子驱动力，建立定量计算模型，以鉴别过程的关键分子基团；最后，在建模结果指导下，通过计算方法去除非必需氨基酸，设计小型化Cas9酶。项目有望建立阐明DNA靶标结合与剪切反应过程基本原理的新理论，揭示过程的关键基团，为Cas9-sgRNA系统的优化改造提供科学认识和计算工具，并开发易于输送的、特异性好的小型化Cas9酶，推进CRISPR技术在生物医学领域的广泛应用。

近年，基于Cas9-sgRNA复合物的CRISPR技术已成为革命性的基因组编辑工具。但是，脱靶效应和低输送效率等问题正严重阻碍该技术的应用。因此，迫切需要研究清楚该复合物靶向切割DNA的分子基础，为系统的优化改造提供科学依据。为解决此问题，本项目首先应用单颗粒冷冻电镜方法解析了SpCas9和SaCas9的Cas9-sgRNA-DNA复合物的结构，获得了它们的DNA剪切活性构象，为阐明Cas9-sgRNA靶向切割DNA链的分子基础提供了关键信息，并为酶的优化改造提供了重要的结构生物化学基础。在此基础上，对SpCas9剪切DNA链的水解机理进行了结构建模，揭示了酶活性中心结合水分子、金属离子Mg2+和DNA链磷酸骨架的关键氨基酸，建立了关于DNA剪切水解反应的机理模型，从原子水平解释了SpCas9如何切断DNA双链。最后，用基于复合物电镜密度的片段删除和定向进化位点重设计的方法，成功地开发了多个小型化、低脱靶和多PAM识别的SpCas9酶新变体。因此，本项目的成果有助于阐明CRISPR-Cas9系统的DNA剪切反应机理，为系统的优化改造和应用提供新的科学认识；而且，项目所得的SpCas9变体有望进一步开发成为易输送、脱靶效应低和效率高的基因编辑系统，提供有价值的基因编辑工具，推进CRISPR技术在生物医学领域的广泛应用。