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

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.