基于CRISPR/Cas9系统的HR和NHEJ修复基因筛选及其调控放射敏感性的功能研究

Ionizing radiation-induced DNA double-strand breaks (DSB) are the critical events of their biological effects. Both homologous recombination (HR) and non-homologous end joining (NHEJ) are the main mechanisms responsible for DSB repair, and they are one of the determining factors of radiosensitivity. CRISPR/Cas9 system is a powerful tool for genome editing, which could the create site-specific DSB. We previously established a CRISPR/Cas9-based approach for detecting NHEJ activity and screened more than 1500 kinds of drugs. Herein, the aim of the current project is to continue making use of the precise targeting of CRISPR/Cas9 system and broaden the detection range and improve the resolution by designing a strategy of exogenous tag DNA import, therefore establish a reliable assay for HR and NHEJ repair activities. Next, we plan to perform siRNA library screening by using this new method and attempt to discover novel genes involved in the HR and NHEJ repair processes. Finally, bioinformatical analysis and molecular and cellular biological experiments would be performed to unveil the functions and mechanisms of these genes in regulating radiosensitivity.

电离辐射诱导的DNA双链断裂（DSB）是其生物学效应的核心事件，同源重组（HR）和非同源末端连接（NHEJ）是负责DSB修复的两种主要机制，是放射敏感性的决定因素之一。CRISPR/Cas9系统是基因组编辑的有力工具，可定点诱导DSB的产生，我们前期成功构建了基于该技术的NHEJ修复活性检测方法，并对1500多种药物进行了筛选。本项目拟继续利用CRISPR/Cas9系统的精准靶向性，通过设计带有标记序列的外源性DNA导入策略，扩展探测范围，提升检测分辨率，建立可定量分析HR和NHEJ修复活性的可靠方法。本项目拟进一步利用该新方法进行siRNA文库的筛选，试图发现参与HR和NHEJ修复过程的新基因，并通过生物信息学分析和分子细胞生物学实验手段揭示其调节放射敏感性的功能和机制。

诱导DNA双链断裂是电离辐射杀伤肿瘤细胞的重要机制，细胞可通过同源重组HR和非同源末端连接NHEJ修复DNA双链断裂，其修复活性是影响放射敏感性的决定因素。本项目建立了以CRISPR/Cas9系统为基础的HR与NHEJ活性检测的筛选方法，进行了FDA药物库与磷酸酶siRNA文库的筛选，发现了具有提高细胞放射敏感性的药物与基因。本项目首先成功构建了靶向HPRT的CRISPR/Cas9系统，并进一步引入具有标签Marker的ssODN与dsODN。ssODN与dsODN可分别通过HR和NHEJ修复进入DSB位点，然后通过PCR定量分析标签Marker在基因组中的含量来检测HR与NHEJ的活性，通过对FDA药物库的筛选，发现了omipalisib可显著抑制DNA双链断裂修复并提高肿瘤细胞的放射敏感性。本项目进一步利用二代测序技术建立了高通量分析HR与NHEJ的方法，通过引入不同分子标签Index的PCR引物，可将多个测序样本进行混合同时测序，借助Axe和CRISPResso软件分析，测序4个样本可以实现100次HR与NHEJ活性的检测，有效提升检测的效率，通过对磷酸酶siRNA文库的筛选，发现了数个对HR与NHEJ修复有抑制作用的潜在磷酸酶靶点。本项目构建的HR与NHEJ修复活性的筛选方法对于发现调控DNA双链断裂的新基因具有重要意义，筛选获得的药物与基因对于临床肿瘤放疗增敏提供了实验基础。