Human T cells were isolated from leukopaks with obtained from STEMCELL Technologies (# 70500.1). T lymphocytes were then purified using the EasySep Human T cell isolation kit (STEMCELL Technologies #17951) and activated with Dynabeads Human T-Activator CD3/CD28 (ThermoFisher #11131D) in X-VIVO 15 medium (Lonza #BP04-744Q) supplemented with human serum (5%:Gemini Bioproducts #100-512), IL-7 (5 ng/ml: Miltenyi Biotec #130-095-367), and IL-15 (5 ng/ml: Miltenyi Biotec #130-095-
760) at a density of 1x10^6 cells per ml.

Nucleofection
After 24 h of T cell activation, CD3/CD28 Dynabeads were magnetically removed, and T cells were nucleofected in P3 buffer (Lonza #V4SP-3096) with ribonucleoprotein (RNP) using a 4D-Nucleofector 96-well unit (Lonza #AAF-1003S). An amount of RNP for one reaction was generated by incubating 60 pmol Cas9 protein (QB3 MacroLab) and 120 pmol sgRNA (Synthego) at 37°C. 2x10^6 cells were electroporated with RNP per well. Lonza program code DN-100 was used for murine T cells, and EH-115 was used for human T cells. After nucleofection, cells were diluted in culture medium and incubated (37°C, 5% CO2). For making knock-ins, AAV was added to the culture between 30–60 min after nucleofection at the indicated MOI, and the culture was incubated overnight. The next day, the medium was exchanged for fresh T cell medium, and cells were expanded using standard culture conditions and maintained at a density of approximately 2x10^6 cells per ml.