利用CRISPR/Cas9技术高通量筛选调控HR修复的lncRNA及其影响辐射敏感性的机制研究

DNA double-strand breaks (DSBs) are the most harmful lesions among DNA damages induced by ionizing radiation, which could be repaired by homologous recombination (HR) and non-homologous end joining(NHEJ). The regulatory functions of long noncoding RNA (lncRNA) in HR repair are considered to be responsible for radiosensitivity and genome stability. In a previous study, we presented a CRISPR/Cas9-based screening for NHEJ inhibitors， which could promote the discovery of novel radiosensitizers. Here, we intend to continue utilizing the site-specific DSB repair model induced by CRISPR/Cas9 system, and import the exogenous double-strand DNA (dsDNA) harbored a unique marker sequence, which serves as the donor template for HR repair, therefore develop a high-throughput detecting approach for HR repair activity. To identify new functions of lncRNA involved in the regulation of HR repair, we attempt to carry out a screening for siRNA library targeting lncRNA. The mechanisms of these lncRNAs in regulating radiosensitivity will be further uncovered, so as to provide creative strategy and novel molecular targets for the research on DNA damage repair caused by ionizing radiation.

DNA双链断裂（DSB）是电离辐射诱导的最严重的DNA损伤类型，主要通过同源重组（HR）和非同源末端连接（NHEJ）两种机制进行修复。长链非编码RNA（lncRNA）对HR修复的调控功能是影响辐射敏感性和基因组稳定性的关键因素。我们前期基于CRISPR/Cas9系统成功建立了针对NHEJ修复的检测方法并应用于辐射增敏药物的筛选研究。本项目拟继续利用CRISPR/Cas9系统定点诱导的DSB损伤修复模型，同时导入含有标记序列的外源性双链DNA作为HR修复模板，构建针对HR修复的高通量检测体系。通过筛选靶向lncRNA的siRNA文库，试图发掘lncRNA参与调控HR修复的新功能，揭示关键lncRNA调节放射敏感性的作用机制，为电离辐射诱导的DNA损伤修复研究提供新的研究思路和分子靶点。

DNA 双链断裂（DSB）是电离辐射生物学效应的核心事件，同源重组修复基因功能是影响真核细胞辐射敏感性和基因组稳定性的关键因素。lncRNA 在HR修复核心分子的调控网络中扮演重要角色。本项目利用前期构建的基于CRISPR/Cas9系统的高通量检测体系，通过筛选靶向lncRNA的siRNA文库，首次发现lncRNA 1664和lncRNA 105377102参与调控HR修复的新功能。研究结果表明：（1）下调lncRNA 1664和lncRNA 105377102表达抑制电离辐射诱导的DSB损伤修复，延长G2/M期阻滞，增加Hela细胞辐射敏感性；（2）下调lncRNA 1664、lncRNA 105377102表达抑制细胞内HR修复，对NHEJ修复水平无明显影响；（3）lncRNA 1664通过调控SIRT1的转录参与调节电离辐射诱导的DSB损伤修复；lncRNA 105377102参与维持HR修复核心蛋白RAD51的mRNA稳定性。本研究为电离辐射诱导DSB修复机制的研究提供新的实验手段，揭示了lncRNA 1664、lncRNA 105377102调控HR修复的新功能，为辐射诱导DSB损伤修复的基础研究提供新的理论依据和分子靶点。