Construction of TRIM21 Knockout Stable Hela Cell Line by CRISPR/Cas9 Technology

doi:10.7668/hbnxb.20195539

Abstract

Abstract:

It aimed to successfully construct a stable human cervical cancer (HeLa) cell line with TRIM21 gene knockout using CRISPR/Cas9 gene editing technology.First,two specific sgRNAs (sgRNA1 and sgRNA2) targeting the TRIM21 gene were designed and synthesized,and they were cloned into the LentiCRISPRv2 vector using PCR amplification and seamless cloning techniques.Subsequently,the editing vectors were transfected into HeLa cells via electroporation,and monoclonal cell lines with successfully integrated editing vectors were obtained through puromycin screening.After validation by Western Blot and expansion of culture,a stable TRIM21-knockout HeLa cell line was established.Based on this cell line,we further investigated the role of TRIM21 in regulating the type I interferon signaling pathway.The experimental results demonstrated that TRIM21 knockout significantly suppressed the expression levels of MDA5,MAVS,and IRF3 in the cells,while also inhibiting the phosphorylation of IRF3,revealing the critical regulatory role of TRIM21 in the type I interferon signaling pathway.The TRIM21-knockout HeLa cell line successfully constructed in this study provides a reliable cellular model for in-depth research into the mechanism of TRIM21 in regulating viral replication and its role in innate immune responses.Furthermore,by optimizing experimental methods,this study confirmed the efficiency and stability of electroporation in constructing gene-knockout cell lines,offering technical references for the construction of other gene-knockout cell lines.

Key words: Cell line, TRIM21, CRISPR/Cas9, Electroporation, Human cervical cancer cell (HeLa)

CLC Number:

S813.3生物技术遗传育种

YANG Dongliang, SHAO Shuai, JI Xiaolan, ZHANG Derong, BAI Jialin, LI Qiongyi. Construction of TRIM21 Knockout Stable Hela Cell Line by CRISPR/Cas9 Technology[J]. Acta Agriculturae Boreali-Sinica, 2025, 40(6): 231-238. doi: 10.7668/hbnxb.20195539.

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http://www.hbnxb.net/EN/Y2025/V40/I6/231

Figures/Tables 5

Fig.1 Sequence of TRIM21-Cas9-sgRNA-LentiCRISPRv2 plasmid A.Sequence of the recombinant TRIM21-Cas9-sgRNA1-LentiCRISPRv2 plasmid; B.Sequence of the recombinant TRIM21-Cas9-sgRNA2-LentiCRISPRv2 plasmid.

Fig.2 Electrophoresis image of PCR products A.PCR amplification product of sgRNA1 (M.DL5000 DNA Marker);B.PCR amplification product of sgRNA2 (M.DL5000 DNA Marker).

Fig.3 TRIM21-sgRNA plasmid double enzyme digestion verification A.Verification of TRIM21-sgRNA1 plasmid by double digestion with Kpn Ⅰ and EcoR Ⅴ(M.1 kb DNA Marker);B.Verification of TRIM21-sgRNA2 plasmid by double digestion with Kpn Ⅰ and EcoR Ⅴ(M.1 kb DNA Marker).

Fig.4 TRIM21 knockout verification A.Five TRIM21 knockout monoclonal cell lines (1—5) post-screening and one negative control monoclonal cell line;B.Western Blot analysis of TRIM21 expression levels in the five TRIM21 knockout monoclonal cell lines;C.Morphology of the TRIM21 KO#1 cell line,TRIM21 NC cell line,and HeLa cell line.Scale bar.100 μm.

Fig.5 TRIM21 knockout functional verification A.Western Blot was used to detect the expression of signal molecules MDA5,MAVS,IRF3 and p-IRF3 in type Ⅰ interferon signaling pathway after TRIM21 knockout,NC was used to extract the total protein of TRIM21 NC cell line,and KO was used to extract the total protein of TRIM21 KO#1 cell line;B—F.Gray analysis of TRIM21,MDA5,MAVS,IRF3 and p-IRF3,different letters indicate significant difference(P<0.001).

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