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Updated: Aug 8, 2025
Electroporation-Based CRISPR-Cas9-Mediated Gene Knockout in THP-1 Cells and Single-Cell Clone Isolation
Published on: February 28, 2025
One-step CRISPR-Cas9-mediated knockout of native TCRαβ genes in human T cells using RNA electroporation
Donovan Flumens1, Diana Campillo-Davo1, Ibo Janssens1
1Laboratory of Experimental Hematology, Vaccine & Infectious Disease Institute (VAXINFECTIO), Faculty of Medicine and Health Sciences, University of Antwerp, 2610 Wilrijk, Belgium.
Abstract:
To avoid mispairing between native and introduced T cell receptors (TCRs) and to prevent graft-versus-host disease in allogeneic T cell therapies, TCRα and TCRβ chains of native TCRs are knocked out via CRISPR-Cas9. We demonstrate the isolation and activation of CD8+ T cells followed by electroporation of T cells with in vitro transcribed eSpCas9(1.1)-P2A-EGFP mRNA and single-guide RNAs targeting the TCRα and TCRβ constant regions. We then describe a flow cytometric analysis to determine TCR knockout efficiency.
Insights
CRISPR-Cas9 gene editing is used to knock out native T cell receptors (TCRs) in CD8+ T cells. This method prevents mispairing and graft-versus-host disease in allogeneic cell therapies.
Area of Science:
- Immunology
- Gene Editing
- Cell Therapy
Background:
- Allogeneic T cell therapies face challenges with native T cell receptor (TCR) mispairing.
- Graft-versus-host disease (GVHD) is a significant risk in these therapies.
Purpose of the Study:
- To develop a method for knocking out native TCRs in CD8+ T cells.
- To prevent TCR mispairing and GVHD in allogeneic T cell therapies.
Main Methods:
- Isolation and activation of CD8+ T cells.
- Electroporation with in vitro transcribed eSpCas9(1.1)-P2A-EGFP mRNA and sgRNAs targeting TCR constant regions.
- Flow cytometry for assessing TCR knockout efficiency.
Main Results:
- Demonstrated successful knockout of TCRα and TCRβ chains.
- Established a method for efficient TCR gene editing in T cells.
Conclusions:
- CRISPR-Cas9 mediated TCR knockout is a viable strategy for enhancing allogeneic T cell therapy safety.
- This approach addresses key barriers to successful T cell transplantation.
