T cells were activated with Dynabeads Human T-Activator CD3/CD28 (Thermo Fisher Scientific) (1:1 beads:cell) in X-VIVO 15 medium (Lonza) supplemented with 5% human serum (Gemini Bioproducts), penicillin–streptomycin (Thermo Fisher Scientific, 50 U/ml), IL-7 (Miltenyi Biotec, 5 ng/ml) and IL-15 (Miltenyi Biotec, 5 ng/ml) and cultured at 10^6 cells/ml. Medium was exchanged every 2–3 days and cells were re-suspended at 10^6 cells ml-1. After 48 hours of T cell activation, cells were detached from CD3/CD28 Dynabeads and the beads were magnetically removed. T cells were electroporated with ribonucleoprotein (RNP) using a 4D-Nucleofector 96-well unit (Lonza). RNP for each electroporation reaction was generated by co-incubating 60 pmol of recombinant Cas9 protein (QB3 MacroLab) with 120 pmol of TRAC sgRNA
(Synthego, CAGGGUUCUGGAUAUCUGU) at 37 °C for 15 min. Cells were re-suspended in P3 primary cell solution (Lonza) (2 × 10^6 live cells per electroporation) and mixed with RNP, followed by electroporation using the EH115 Nucleofector protocol. Cells were then diluted into serum-free medium (2 × 10^6 cells/ml) and incubated at 37 °C and 5% CO2.