Construct design and validation for transduction of primary T cells. (A) Schematic representation of transgenes cloned into the pELNS vector (top) or lentiCRISPRv2 vector (bottom). (B) Timeline for transduction and selection of primary T cells. (C) Graphical representation of TCR expression on primary T cells transduced with pELNS vector, with and without lentiCRISPRv2 vector (top). Gray molecules represent endogenous TCR chains whereas blue ones represent transduced TCR chains. The histograms below show endogenous αβ TCR expression in 3 donors (gray, untransduced; blue, transduced only with a γδ TCRl red, transduced with a γδ TCR and CRISPR), as well as a representative unstained control (black). The color coding is maintained throughout the manuscript. The numbers on histograms refer to geometric mean fluorescence intensities of αβ TCR expression across 3 donors (D1, D2, D3). EF-1α, elongation factor-1 α promoter; EFS, short EF-1α promoter; pac, puromycin N-acetyltransferase; U6, RNA polymerase III promoter.