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

To investigate the cellular target selectivity of small molecules targeting thioredoxin reductase 1, we reported the construction and functional research of a stable gene (encode thioredoxin reductase 1) knockout HCT-116 cell line. We designed and selected knockout sites according to the gene sequence and CRISPR/Cas9 target designing principles. SgRNA oligos based on the selected 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, knockout HCT-116 cells were selected using puromycin resistance. The 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 gene within HCT-116 cells, and no expression of TrxR1 protein could be observed in stable knockout HCT-116 (HCT116--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 gene knockout HCT-116 cell line was obtained through CRISPR/Cas9 techniques, which may facilitate investigating the role of TrxR1 in various diseases.