CRISPR/Cas9-mediated Gene Knockout Followed by Negative Selection Leads to a Complete TCR Depletion in orthoCAR19 T Cells

Genome-editing technologies, especially CRISPR (clustered regularly interspaced short palindrome repeats)/Cas9 (CRISPR-associated protein 9), endows researchers the ability to make efficient, simple, and precise genomic DNA changes in many eukaryotic cell types. CRISPR/Cas9-mediated efficient gene knockout holds huge potential to improve the efficacy and safety of chimeric antigen receptor (CAR) T cell-based immunotherapies. Here, we describe an optimized approach for a complete loss of endogenous T cell receptor (TCR) protein expression, by CRISPR/Cas9-mediated TCR alpha constant (TRAC) and TCR beta constant (TRBC) gene knockout, followed by subsequent CD3 negative selection in engineered human orthoCAR19 T cells. We believe this method can be expanded beyond CAR T cell application, and target other cell surface receptors.