One-step CRISPR-Cas9-mediated knockout of native TCRαβ genes in human T cells using RNA electroporation

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.

Keywords: CRISPR; Cell culture; Cell isolation; Flow Cytometry/Mass Cytometry; Immunology; Tissue Engineering.