CRISPR/Cas9-based knockout library screening identifies BAG3 as a potential regulator of radiosensitivity
10.7644/j.issn.1674-9960.2025.06.004
- VernacularTitle: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
- Author:
Zhengyue CAO
1
;
Youfeng ZHANG
;
Zhichun LÜ
;
Huiying GAO
;
Shensi XIANG
;
Jingjing LI
;
Xiaofei ZHENG
;
Changyan LI
Author Information
1. 军事科学院军事医学研究院,北京 100850
- Keywords:
clustered regularly interspaced short palindromic repeats(CRISPR);
CRISPR-associated nuclease 9(Cas9);
colon cancer;
gene knockout;
Bcl2-associated athanogene 3
- From:
Military Medical Sciences
2025;49(6):421-429
- CountryChina
- Language:Chinese
-
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.
