广西医科大学学报2025，Vol.42Issue(2)：241-246,6.DOI:10.16190/j.cnki.45-1211/r.2025.02.011
利用CRISPR/Cas9在HEK293T细胞实现γ珠蛋白上调
CRISPR/Cas9-mediated γ-globin expression upregulation in HEK293T cells
摘要
Abstract
Objective:To edit the γ-globin promoter of HEK293T cells using the CRISPR/Cas9 technology to upregulate γ-globin and explore the feasibility of using gene editing technology for the treatment of β-thalasse-mia.Methods:Using gene editing technology to treat β-thalassemia,two sgRNA sequences were designed at 420 bp upstream of the γ-globin transcription start site(TSS).CRISPR/Cas9 plasmids were transfected via lipo-fectamine to induce cleavage of the hemoglobin gamma(HBG)gene promoter in HEK293T cells.Positive clone screening,Sanger sequencing validation,TIDE analysis for editing efficiency,reverse transcription-quantitative polymerase chain reaction(RT-qPCR)and western blotting were used to detect the expression level of the γ-glo-bin gene.Results:Both sgRNAs achieved efficient gene cutting on γ-globin,with cutting of 44.20%(sgRNA1)and 32.90%(sgRNA2),respectively.The results of RT-qPCR and western blotting showed that,compared to the negative control groups(NCs transfected with empty PX459 plasmid),the expression levels of the γ-globin gene in the sgRNA2 group were elevated(P＜0.05).Conclusion:The CRISPR/Cas9 gene editing technology can achieve effective editing on the γ-globin promoter,thereby upregulating γ-globin.关键词
CRISPR/Cas9编辑/γ珠蛋白/HEK293T细胞/基因编辑Key words
CRISPR/Cas9 editing/γ-globin/HEK293T cells/gene editing分类
医药卫生引用本文复制引用
黄献娟,赖永榕,李静..利用CRISPR/Cas9在HEK293T细胞实现γ珠蛋白上调[J].广西医科大学学报,2025,42(2):241-246,6.基金项目
国家卫生健康委员会人才交流中心科研课题资助项目(No.RCLX2315013) （No.RCLX2315013）
广西自然科学基金资助项目(No.305094881019) （No.305094881019）
