生物技术通报2025，Vol.41Issue(5)：52-61,10.DOI:10.13560/j.cnki.biotech.bull.1985.2024-1033
具备高效CRISPR协同激活活性的HIEC6-dCas9-SAM稳转细胞株构建
Construction of HIEC6-dCas9-SAM Transgenic Cell Line with Highly-efficient CRISPR Synergistic Activation Properties
摘要
Abstract
[Objective]This study aims to establish HIEC6-dCas9-SAM monoclonal cell lines with high-level transcriptional activating activity using human normal intestinal epithelial cells(HIEC6)as a model,which will serve as valuable tools for utilizing the CRISPR activation(CRISPRa)system to screen key genes involved in the pathogenesis of human intestinal diseases and to explore the underlying molecular mechanisms.[Method]HIEC6-dCas9-SAM polyclonal cells were first constructed using the PiggyBac transposon system.Monoclonal cell lines were then selected using the limited dilution method,and the expressions of dCas9-SAM proteins(dCas9,VP64,MS2,HSF1,and p65)in these monoclonal cell lines was confirmed by Western blotting and indirect immunofluorescence.Finally,CRISPRa fluorescence reporting system and lentivirus-based sgRNA constructs targeting specific genes were employed to evaluate the CRISPRa efficiency of the monoclonal cell lines at both the transcriptional and protein levels.[Result]Two HIEC6-dCas9-SAM monoclonal cell lines were successfully generated,stably expressing high levels of dCas9-SAM proteins.The CRISPRa fluorescence reporting system demonstrated activation efficiencies of 96.7%and 99.0%for the two cell lines,respectively.Verification of CRISPRa for the target genes revealed that:The transcriptional activation levels of the APN gene in the two HIEC6-dCas9-SAM monoclonal cell lines were as high as 2 725-fold and 4 521-fold respectively,while those of the SLC35A1 gene were 27.5-fold and 18.1-fold,respectively.At the protein level,the activation efficiencies of APN protein were 12.9-fold and 11.2-fold,respectively,while those of SLC35A1 protein were 1.32-fold and 0.97-fold,respectively.Both monoclonal stable cell lines showed high transcriptional activation activity.[Conclusion]Two HIEC6-dCas9-SAM monoclonal cell lines with highly-efficient CRISPR synergistic activation properties have been successfully established.These cell lines serve as valuable tools for the subsequent screening of key genes involved in the pathogenesis of human intestinal diseases and for exploring the molecular mechanisms underlying these diseases using the CRISPRa system.关键词
dCas9-SAM/HIEC6细胞/CRISPR激活/稳转细胞株Key words
dCas9-SAM/HIEC6 cells/CRISPR activation/stable cell line引用本文复制引用
任柱平,杨泰然,雷元三,金留飞,崔古贞,田益明,陈峥宏..具备高效CRISPR协同激活活性的HIEC6-dCas9-SAM稳转细胞株构建[J].生物技术通报,2025,41(5):52-61,10.基金项目
贵州省科技计划项目(黔科合基础-ZK[2023]一般333),贵州医科大学引进博士启动基金项目(校博合J字[2022]010),贵州省病原生物学重点实验室(QJJ[2022]019),高等学校学科创新引智计划(111计划,D20009) （黔科合基础-ZK[2023]一般333）
