文章引用说明 更多>> (返回到该文章)

Jinek, M., East, A., Cheng, A., Lin, S., Ma, E. and Doudna, J. (2013) Rna-Programmed Genome Editing in Human Cells. Elife, 2, e00471.
http://dx.doi.org/10.7554/elife.00471

被以下文章引用:

  • 标题: SpCas9结构域相互作用关键氨基酸的动态网络分析Dynamical Network Analysis of Key Amino Acids for Domain Interactions of SpCas9

    作者: 李干, 王国栋, 怀聪, 黄强

    关键字: 基因编辑, CRISPR-Cas9, 结构域相互作用, 分子动力学模拟, 动态网络分析Gene Editing, CRISPR-Cas9, Domain Interaction, Molecular Dynamics Simulation, Dynamical Network Analysis

    期刊名称: 《Hans Journal of Computational Biology》, Vol.6 No.3, 2016-09-29

    摘要: CRISPR-Cas9系统是一种操作简单、灵活性好、识别和切割效率高的基因编辑技术,具有用于基因治疗的潜能。然而,由于该系统的主要组分Streptococcus pyogenes Cas9 (SpCas9)蛋白过大,难于向靶细胞运输,因而亟需构建截短体蛋白,以提高系统的运输效率、扩展其应用前景。前期结构研究表明,SpCas9结构域分界清晰,各结构域功能相对独立又彼此协同,易于通过对各结构域进行分步改造实现蛋白截短优化。但报道的晶体结构缺乏SpCas9的全长结构,及结构域间相互作用的关键信息。针对此问题,本文运用分子动力学(Molecular Dynamics, MD)模拟和动态网络分析方法研究SpCas9-sgRNA-DNA复合物结构。我们首先对已有晶体结构的缺失片段进行补齐,构建了一个包含全长SpCas9及DNA链的三元复合物原子结构。然后对复合物结构进行长时间尺度MD模拟,通过动态网络分析方法分析了SpCas9蛋白各结构域间的相互作用,找出影响SpCas9结构域间相互作用的关键氨基酸。与已有实验结果比较,所识别的位点是SpCas9蛋白正常工作的关键氨基酸。因此,本研究不仅可以加深人们对SpCas9作用机制的了解,也为SpCas9蛋白的优化改造提供理论指导。 CRISPR-Cas9 system is widely used in gene editing because of its simple operation, flexibility, and high recognition and cleavage efficiency, and it also has the potential for usage in gene therapy. However, Streptococcus pyogenes Cas9 (SpCas9), the main component in the powerful system is too large to be transferred to target cells, which limits its application. So it is necessary to shorten the length of SpCas9. Previous studies revealed that SpCas9 executed its functions by several cooperate but relatively independent domains, so it is possible to optimize this protein section by section. Nevertheless, the integrated structure of SpCas9 and the information on domain interacting is still lacking in reported crystal structures. To solve this problem, this study analyzed the structure of SpCas9-sgRNA-DNA complex using molecular dynamics (MD) simulation and dynamical network analysis. Firstly, the missing fragment of the existing crystal structure was completed to construct a ternary complex atomic structure containing full-length SpCas9 and non-complemen- tary DNA strand. And then, after a long time MD simulation of this new structure, the interactions between domains of SpCas9 were analyzed by dynamical network analysis. Finally, we determined the key residues for SpCas9 domains to interact with each other. Compared with previous experimental results, the identified sites are critical amino acids for proper SpCas9 function. Thus, this study not only deepens the understanding of the working mechanisms of the CRIPSR-Cas9 system, but also offers important guidelines for the optimization and improvement of the system.

在线客服:
对外合作:
联系方式:400-6379-560
投诉建议:feedback@hanspub.org
客服号

人工客服,优惠资讯,稿件咨询
公众号

科技前沿与学术知识分享