基于结构的CRISPR蛋白xCas9的优化设计

Structure-based optimization and design of CRISPR protein xCas9

摘要:

酿脓链球菌 (Streptococcus pyogenes Cas9，SpCas9) 已成为强大的基因组编辑工具，但其可识别的前间隔序列临近基序 (Protospacer adjacent motifs，PAMs) 范围有限，且存在脱靶效应。为解决这些问题，文中提出一种对SpCas9的定向进化突变体xCas9进行优化的理性方法。首先，使用Rosetta程序进行能量最小化以优化Cas9的三维结构，获得其能量最低的构象；然后，对其定向进化所得氨基酸位点进行组合突变设计；最后，通过自由能排序从设计突变体中筛选出用于实验验证的最优变体。经DNA剪切实验验证，成功地获得了一个多PAM识别和低脱靶的新变体yCas9 (262A/324R/409N/480K/543D/694L/1219T)。该变体可识别NG、GAA和GAT序列，且其由错配sgRNA引导的脱靶DNA剪切活性低，为生物医学领域提供了一个有潜在应用价值的基因编辑工具。同时，文中还对SpCas9、xCas9和yCas9进行了分子动力学模拟，揭示了其PAM识别和脱靶效应的机理，可为进一步的CRISPR/Cas9蛋白优化改造提供理论指导。

Abstract:

Streptococcus pyogenes Cas9 (SpCas9) has become a powerful genome editing tool, but has a limited range of recognizable protospacer adjacent motifs (PAMs) and shows off-target effects. To address these issues, we present a rational approach to optimize the xCas9 mutant derived from SpCas9 by directed evolution. Firstly, energy minimization with the Rosetta program was applied to optimize the three-dimensional structure of Cas9 to obtain the lowest energy conformation. Subsequently, combinatorial mutations were designed based on the mutations sites of xCas9 acquired during the directed evolution. Finally, optimal mutants were selected from the designed mutants by free energy ranking and subjected to experimental verification. A new mutant yCas9 (262A/324R/409N/480K/543D/694L/1219T) with multiple PAM recognition ability and low off-target effects was obtained and verified by DNA cleavage experiments. This mutant recognizes the NG, GAA and GAT PAMs and shows low off-target DNA cleavage activity guided by mismatched sgRNA, thus provides a gene editing tool with potential applications in biomedical field. Furthermore, we performed molecular dynamics simulations on the structures of SpCas9, xCas9 and yCas9 to reveal the mechanisms of their PAM recognition and off-target effects. These may provide theoretical guidance for further optimization and modification of CRISPR/Cas9 proteins.