Journal of Shanghai Jiao Tong University (Medical Science) >

2022 , Vol. 42 >Issue 4: 433 - 442

DOI: https://doi.org/10.3969/j.issn.1674-8115.2022.04.005

Innovative research team achievement column

Application of CRISPR/Cas9-mediated gene editing system to studying the regulation of T-bet in B cells

  • Xiaxia HAN ,
  • Shuangshuang GU ,
  • Dai DAI ,
  • Nan SHEN
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  • Department of Rheumatology, Renji Hospital, Shanghai Jiao Tong University School of Medicine; Shanghai Institute of Rheumatology, Shanghai 200127, China
SHEN Nan, E-mail: nanshensibs@gmail.com.

Received date: 2022-01-24

  Accepted date: 2022-04-25

  Online published: 2022-04-28

Supported by

National Natural Science Foundation of China(31630021);Innovative Research Team of High-Level Local Universities in Shanghai(SSMU-ZDCX20180100)

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Abstract

Objective

·To study the regulation of transcription factor T-bet in antibody isotype switching via the optimized CRISPR/Cas9-mediated gene editing system in primary B cells.

Methods

·In order to demonstrate the regulation of T-bet in antibody isotype switching, the transcriptome data of in vitro-derived B cells from public database GEO was analyzed. The retroviral small guide RNA (sgRNA) vector was constructed to deliver sgRNA targeting Tbx21 (T-box 21, encoding T-bet), and retroviral packaging cell line Plat-E was used for retrovirus production. The retroviruses with sg-Tbx21 plasmids were harvested and concentrated. Primary B cells isolated from Cas9 transgenic mice were activated in vitro by synergistic stimulation of anti-B cell receptor antibody, anti-CD40 antibody and a Toll-like receptor agonist, and then infected with concentrated retroviruses with sg-Tbx21. The genome DNA was extracted from the infected cells isolated by flow cytometry, and then PCR amplification was performed for target gene sequence. ICE CRISPR analysis tool was used to calculate the gene knockout efficiency. The edited B cells were induced to plasma cells in vitro and the antibody isotypes of IgM, IgG2c, and IgG3 production in the culture supernatant were assessed by ELISA. Independent sample t-test was used to analyze the difference of antibody levels in the supernatant between the sg-NC editing cells and sg-Tbx21 editing cells.

Results

·① According to the analysis of GEO database, interferon-γ induced the expression of Tbx21 in B cells, and T-bet expression promoted antibody isotype switching-related pathways. ② The retroviral infection efficiency was improved in primary B cells after in vitro activation, which were infected by the concentrated high titer viruses. ③ The sg-Tbx21 plasmids were delivered to Cas9 transgenic B cells by retroviruses, which led to T-bet knockout efficiency of about 59%. ④ Cultured in the plasma cells-polarizing condition, the Tbx21 edited B cells secreted less IgG2c into the culture supernatant (P=0.000), while the levels of IgM and IgG3 did not significantly change.

Conclusion

·The efficiency of sgRNA delivery can be improved by concentrating retroviruses and activating primary B cells through synergistic stimulation, and Tbx21 gene can be knocked out in primary B cells of Cas9 transgenic mice; it is proved that T-bet knockout hinders the production of antibody isotype IgG2c through this system.

Key words： small guide RNA; retrovirus; B cell; gene editing; T-bet; antibody isotype switching

Cite this article

Xiaxia HAN , Shuangshuang GU , Dai DAI , Nan SHEN . Application of CRISPR/Cas9-mediated gene editing system to studying the regulation of T-bet in B cells[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2022 , 42(4) : 433 -442 . DOI: 10.3969/j.issn.1674-8115.2022.04.005

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