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Protocol for genetic dissection of class switch recombination using genome-editing tools.

Pengfei Dai1, Tingting Liu2, Dingpeng Yang2

  • 1Key Laboratory of RNA Innovation, Science and Engineering, Shanghai Academy of Natural Sciences (SANS), Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai 200031, China; International Peace Maternity and Child Health Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai Key Laboratory of Embryo Original Diseases, Shanghai 200030, China.

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|June 7, 2025
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Summary

This study introduces a new protocol for inducing antibody class switch recombination using base editors or CRISPR-Cas9. This method helps researchers explore DNA metabolic factors in antibody diversification.

Keywords:
CRISPRcell-based assaysimmunology

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Area of Science:

  • Immunology
  • Molecular Biology
  • Genetics

Background:

  • Antibody class switch recombination (CSR) is a crucial process for adaptive immunity.
  • CSR involves programmed DNA damage and requires coordinated action of DNA metabolic factors.
  • Understanding the factors involved in CSR is essential for immune system research.

Purpose of the Study:

  • To present a novel protocol for inducing CSR in B cells.
  • To enable investigation into the roles of specific DNA metabolic factors in CSR.
  • To provide optimized reagents and methods for CSR induction and analysis.

Main Methods:

  • Utilizing base editors or CRISPR-Cas9 for targeted DNA modification.
  • Optimizing guide RNA (gRNA) sequences for efficient gene editing.
  • Implementing cytokine activation, electroporation, and surface immunoglobulin detection.
  • Establishing a framework for data analysis of CSR.

Main Results:

  • Successful induction of class switch recombination using the described protocol.
  • Demonstration of optimized gRNA sequences for base editors and CRISPR-Cas9.
  • Detailed steps for experimental execution and analysis provided.

Conclusions:

  • The presented protocol offers a robust method for studying CSR.
  • This technique facilitates the investigation of DNA metabolic factors in antibody diversification.
  • Researchers can now more effectively elucidate the functional roles of these factors in the immune response.