基于R-loop靶向编辑技术的R-loop功能位点高通量筛选系统  

High-throughput Screening System for Functional R-loop Loci Based on R-loop Targeted Editing Technology

作　　者：皮一飞 宋新辉 王淅琳 李谨谨 孙长斌 徐炜 PI Yi-fei;SONG Xin-hui;WANG Xi-lin;LI Jin-jin;SUN Chang-bin;XU Wei(Shenzhen Branch,Guangdong Laboratory of Lingnan Modern Agriculture,Agricultural Genomics Institute at Shenzhen,Chinese Academy of Agricultural Sciences,Shenzhen 518000;School of Life Sciences,Henan University,Kaifeng 475001;College of Animal Science and Technology,Guangxi University,Nanning 530004)

机构地区：[1]中国农业科学院深圳农业基因组研究所(岭南现代农业科学与技术广东省实验室深圳分中心),深圳518000 [2]河南大学生命科学学院,开封475001 [3]广西大学动物科学技术学院,南宁530004

出　　处：《生物技术通报》2024年第10期181-190,共10页Biotechnology Bulletin

基　　金：国家重点研发计划(2021YFF1000600);中国农业科学院青年创新专项(Y2022QC33);国家自然科学基金项目(32071437,32100423)。

摘　　要：【目的】开发哺乳动物细胞R-loop靶向编辑技术,探究其在肿瘤细胞耐药性研究中的应用。【方法】构建无酶切活性的Cas9与具有R-loop水解活性的RNase H1融合蛋白dCas9-RNaseH1表达载体,用于实现对R-loop的靶向编辑。在HeLa细胞中稳定表达该融合蛋白,建立R-loop靶向编辑细胞模型。转染覆盖全基因组转录起始区域的sgRNA文库,构建R-loop筛选细胞库,筛选影响紫杉醇和顺铂耐药性的R-loop功能位点。【结果】筛选获得744个影响HeLa细胞耐药性的R-loop功能位点,覆盖了细胞周期、凋亡、信号传导等关键生物通路。其中,26个位点使HeLa细胞对这两种药物产生耐药性,8个位点使其对这两种药物敏感,提示可能存在共享的生物通路。功能验证显示,部分R-loop功能位点通过调节相关基因(如ZBTB20、SPON2、ACTRT1等)的表达来影响HeLa细胞对抗肿瘤药物的敏感性。【结论】成功开发出适用于哺乳动物细胞的R-loop靶向编辑系统,并建立高通量筛选平台。【Objective】To develope R-loop targeted editing technology for mammalian cells and explore its applications in drug resistance of tumor cells.【Method】An expression vector was constructed to express a dCas9-RNaseH1 chimaera that combines catalytically dead Cas9 lacking endonuclease activity with RNase H1 possessing R-loop hydrolysis activity for R-loop targeted editing.This dCas9-RNaseH1 chimaera was transfected to HeLa cells and stably expressed to construct a model cell line.To create a cell library for R-loop screening,a genome-wide gRNA library covering transcription start sites was transfected to the model cell line,through which R-loop functional sites affecting resistance to paclitaxel and cisplatin were identified.【Result】Total 744 R-loop functional sites affecting HeLa cell drug resistance were identified,which cover key biological pathways such as cell cycle,apoptosis,and signal transduction.Among them,26 sites confered resistance to two drugs,while 8 sites rendered sensitivity to both drugs,suggesting potential shared biological pathways.Functional validation revealed that certain R-loop sites modulated the expressions of relevant genes(e.g.,ZBTB20,SPON2,ACTRT1),significantly impacting HeLa cell sensitivity to anticancer drugs.【Conclusion】A R-loop targeted editing system is successfully developed and a high-throughput screening platform is established for mammalian cells.

关 键 词：高通量筛选 耐药性 R-loop 定点调控 CRISPR/dCas9 

分 类 号：Q78[生物学—分子生物学]