一种用于任意序列DNA和RNA高效编辑的HpSGN-neo系统

针对现有基因编辑工具酶不能高效编辑任意序列DNA和RNA底物的问题，本项目将在自主构建的第一、二代结构介导基因编辑工具（SGN）基础上，通过设计具有发卡二级结构的向导RNA探针Hp-gRNA，构建具有识别RNA探针和切割核酸底物功能的重组酶Cle-Edit，开发出第三代SGN系统，HpSGN-neo。其中，Hp-gRNA在核内呈高浓度状态，与Cle-Edit形成复合物整体，以实现“高效”；Cle-Edit可区分单碱基差异，以实现“精准”；Cle-Edit氨基酸个数较少，以实现“小巧”；HpSGN-neo与靶标结合由核酸结构介导，没有序列偏爱性，以实现“无序列限制”，并且还可同时编辑DNA和RNA。本课题将在体外和细胞水平对HpSGN-neo的编辑效能进行研究优化，并将其应用于小鼠体内癌细胞单碱基突变位点的靶向敲击，同时与传统基因编辑工具比较评估。为基因编辑技术的研发提供了新思路。

Aiming at the problem that the present gene editing tools can not edit DNA and RNA without sequence limitation, this project will develop the 3rd generation SGN system, HpSGN-neo system. By designing the Hp-gRNA, a guide RNA probe with secondary structure of hairpin, and constructing the Cle-Edit, a recombinant enzyme with the function of recognizing RNA probe and cutting nucleic substrate，the HpSGN-neo system is developed. Firstly, the Hp-gRNA is in a high concentration state in the nucleus and forms a complex with Cle-Edit, which is to achieve "high efficiency". Secondly, the Cle-Edit can distinguish single base differences to achieve "accuracy". Thirdly, the Cle-Edit has a small number of amino acids to achieve "small size". Fourthly, the HpSGN-neo system’s capture is mediated by nuclear acid structure without sequence preference, which is to achieve "no sequence restriction", and it can also cleave both DNA and RNA substrate. This project will study and optimize the editing ability of the HpSGN-neo system in vitro and in cellular, then apply it in vivo to achieve the single mutate base editng. The HpSGN-neo system will be compared with the present gene editing tools. It provides a new idea for the research and development of gene editing technology.