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

Zhiyin Zhou; Xiaomei LÜ; Li Zhu; Ji Zhou; Huidan Huang; Chao Zhang; Xiaoping Liu

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

Author: Zhiyin ZHOU ¹ ; Xiaomei LÜ ¹ ; Li ZHU ¹ ; Ji ZHOU ² ; Huidan HUANG ¹ ; Chao ZHANG ¹ ; Xiaoping LIU ¹
Author Information

1. Center of Drug Screening and Evaluation, Wannan Medical College, Wuhu 241000, Anhui, China.
2. Center of Reproductive Medicine, The First Affiliated Hospital of Wannan Medical College, Wuhu 241000, Anhui, China.
Publication Type:Journal Article
Keywords: CRISPR/Cas9; TrxR1; gene knockout; stable cell line
MeSH: CRISPR-Cas Systems/genetics*; Gene Editing; Gene Knockout Techniques; HCT116 Cells; Humans; RNA, Guide/metabolism*
From: Chinese Journal of Biotechnology 2022;38(3):1074-1085
CountryChina
Language:Chinese
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.