CRISPR/Cas9-mediated Gene Knockout Followed by Negative Selection Leads to a Complete TCR Depletion in ortho CAR19 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 α constant (TRAC) and TCR β constant (TRBC) gene knockout, followed by subsequent CD3 negative selection in engineered human ortho CAR19 T cells. We believe this method can be expanded beyond CAR T cell application, and target other cell surface receptors. Graphical abstract: Schematic overview of the two-step process of endogenous TCR depletion in engineered human ortho CAR19 T cells using (1) CRISPR/Cas9-mediated gene knockout followed by (2) CD3 negative selection.