Genome Engineering of Primary Human B Cells Using CRISPR/Cas9

Area of Science:

• Immunology
• Molecular Biology
• Biotechnology

Background:

• B cells are crucial lymphocytes for adaptive immunity, offering potential for cell-based therapies due to their accessibility and antibody production capabilities.
• Their natural function of producing antibodies can be harnessed to express therapeutic proteins, addressing infections and genetic disorders.

Purpose of the Study:

• To establish a comprehensive protocol for engineering primary human B cells.
• To enable gene knockout and transgene integration for studying gene function and developing novel B cell therapeutics.

Main Methods:

• Isolation and in vitro activation/expansion of primary human B cells from peripheral blood mononuclear cells (PBMCs).
• Application of CRISPR/Cas9 for site-specific gene knockout (KO) in B cells.
• Utilizing CRISPR/Cas9 with recombinant adeno-associated viral (rAAV) vectors for precise transgene integration.

Main Results:

• Demonstrated efficient gene KO in primary human B cells using CRISPR/Cas9.
• Successfully integrated transgene expression cassettes site-specifically into B cells via CRISPR/Cas9 and rAAV vectors.
• Developed a robust, step-by-step platform for B cell engineering.

Conclusions:

• The presented protocol provides a versatile platform for advancing B cell research and therapeutic development.
• This engineering approach facilitates the study of gene functions and the creation of novel B cell-based therapies.