Protocol One-step CRISPR-Cas9-mediated knockout of native TCRa13 genes in human T cells using RNA

To avoid mispairing between native and introduced T cell receptors (TCRs) and to prevent graft-versus-host disease in allogeneic T cell therapies, TCRa and TCR13 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 TCRa and TCR13 constant regions. We then describe a flow cytometric analysis to determine TCR knockout efficiency.