军事医学2025，Vol.49Issue(6)：421-429,9.DOI:10.7644/j.issn.1674-9960.2025.06.004
利用CRISPR/Cas9敲除文库筛选发现一种新的辐射敏感性调控基因BAG3
CRISPR/Cas9-based knockout library screening identifies BAG3 as a potential regulator of radiosensitivity
摘要
Abstract
Objective To identify genetic regulators of ionizing radiation(IR)sensitivity through clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated nuclease 9(Cas9)genome-wide screening.Methods A specialized single guide RNA(sgRNA)library was developed targeting 987 stress-response regulatory genes identified through Kyoto Encyclopedia of Genes and Genomes(KEGG),Reactome and gene ontology(GO)databases,followed by construction of sgRNA plasmids and packaging into a lentiviral library.Using colon adenocarcinoma Caco-2 cells as a model system,the Cas9-stable transgenic line was established via lentiviral transduction and antibiotic selection.Library-transduced cells underwent puromycin selection,followed by γ-irradiation(dose optimized via preliminary experiments).Post-irradiation phenotypic selection was conducted after 14 days,with subsequent next-generation sequencing of surviving cell populations to identify enriched/depleted sgRNAs.Candidate genes were functionally validated through orthogonal assays:cell counting kit-8(CCK-8)proliferation analysis,clonogenic survival assays and flow cytometric quantification of reactive oxygen species(ROS)and apoptotic markers.Results The optimized screening platform identified 281 radiation response genes(165 radiosensitive and 116 radioprotective candidates).Functional validation revealed Bcl2-associated athanogene 3(BAG3)knockdown significantly enhanced radioresistance.Proliferation was increased 1.2-1.5 fold,clonogenic survival improved 1.5-2.0 fold,and ROS was reduced by 13％-25％coupled with 32％-73％apoptosis attenuation compared to control groups.Conclusion The integrated CRISPR/Cas9 screening platform effectively identifies novel radiation response regulators,as evidenced by the discovery of BAG3 as a critical radiosensitivity determinant.This high-throughput functional genomics approach provides a robust methodology for systematically mapping molecular determinants of cellular radiation response,with potential applications in both mechanistic studies and therapeutic target discovery.关键词
规律成簇间隔短回文重复/CRISPR相关核酸酶9/结肠癌/基因敲除/Bcl2相关永生基因3Key words
clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated nuclease 9(Cas9)/colon cancer/gene knockout/Bcl2-associated athanogene 3分类
生物科学引用本文复制引用
曹正岳,张友凤,吕志春,高慧英,向慎思,李晶晶,郑晓飞,李长燕..利用CRISPR/Cas9敲除文库筛选发现一种新的辐射敏感性调控基因BAG3[J].军事医学,2025,49(6):421-429,9.基金项目
国家重点研发计划(2022YFA1103502) （2022YFA1103502）
