利用CRISPR/Cas9系统构建稳定敲除TrxR1基因的HCT-116细胞株

Construction of a stable TrxR1 knockout HCT-116 cell line using CRISPR/Cas9 gene editing system

摘要:

为更好地研究靶向硫氧还蛋白还原酶1的小分子化合物的细胞内靶点选择性，利用CRISPR/Cas9系统构建稳定敲除TrxR1基因(编码硫氧还蛋白还原酶1)的HCT-116细胞株。首先根据TrxR1基因序列和CRISPR/Cas9靶点设计原则，设计并选择合适的敲除位点，再根据敲除位点序列设计敲除TrxR1基因的sgRNA干扰序列，以pCasCMV-Puro-U6空质粒载体为骨架构建能表达该sgRNA干扰序列的重组质粒。质粒共转染至HCT-116细胞后，利用嘌呤霉素筛选TrxR1敲除的HCT-116细胞，通过DNA测序、免疫蛋白印迹、TRFS-green荧光探针和细胞内TrxR1酶活力检测等方法鉴定和验证HCT-116细胞的TrxR1基因敲除效果。进一步通过CCK-8实验初步研究靶向TrxR1小分子化合物对细胞内TrxR1酶活力和细胞增殖力抑制的相关性。结果显示，表达sgRNA干扰序列的重组质粒可以敲除HCT-116细胞中TrxR1基因，筛选获得的稳定敲除细胞HCT116-TrxR1-KO中无TrxR1蛋白表达，而靶向TrxR1小分子抑制剂对该细胞无TrxR1酶活力和细胞增殖力抑制效果。本研究利用CRISPR/Cas9系统成功构建了HCT-116的TrxR1基因敲除的稳定细胞株，为进一步研究TrxR1在相关疾病的发生机制和治疗中的作用奠定了基础。

Abstract:

To investigate the cellular target selectivity of small molecules targeting thioredoxin reductase 1,we reported the construction and functional research of a stable TrxR1 gene (encode thioredoxin reductase 1) knockout HCT-116 cell line.We designed and selected TrxR1 knockout sites according to the TrxR1 gene sequence and CRISPR/Cas9 target designing principles.SgRNA oligos based on the selected TrxR1 knockout sites were obtained.Next,we constructed knockout plasmid by cloning the sgRNA into the pCasCMV-Puro-U6 vector.After transfection of the plasmid into HCT-116 cells,TrxR1 knockout HCT-116 cells were selected using puromycin resistance.The TrxR1 knockout efficiency was identified and verified by DNA sequencing,immunoblotting,TRFS-green fluorescent probe,and cellular TrxR1 enzyme activity detection.Finally,the correlation between TrxR1 expression and cellular effects of drugs specifically targeting TrxR1 was investigated by CCK-8 assay.The results demonstrated that the knockout plasmid expressing the sgRNA effectively knocked-out TrxR1 gene within HCT-116 cells,and no expression of TrxR1 protein could be observed in stable TrxR1 knockout HCT-116(HCT116-TrxR1-KO) cells.The TrxR1-targeting inhibitor auranofin did not show any inhibitory activity against either cellular TrxR1 enzyme activity or cell proliferation.Based on these results,we conclude that a stable TrxR1 gene knockout HCT-116 cell line was obtained through CRISPR/Cas9 techniques,which may facilitate investigating the role of TrxR1 in various diseases.