Isolation and Culture of Primary Human T Cells
T cell isolation was done as previously described (Roth et al., 2020). Briefly, human T cells were isolated from leukapheresis products (Leukopaks, Stemcell, samples collected with approved Stemcell IRB) or TRIMA Apheresis (Blood Centers of the Pacific, San Francisco) using EasySep Human T Cell Isolation Kit (Stemcell). The use of human material is approved by the UCSF Committee on Human Research (CHR #13-11950). T cells were cultured in X-VIVO 15 media (Lonza Bioscience) supplemented with 5% fetal bovine serum (FBS), 50 µM 2mercaptoethanol (Thermo Fisher Scientific), and 10 mM N-Acetyl-L-Cysteine (VWR). Prior to electroporation, T cells were stimulated for 48 hours at 10^6 cells per ml of media containing 500 U/ml IL-2 (R&D Systems), 5 ng/ml IL-7 (R&D Systems), 5 ng/ml IL-15 (R&D Systems), and CTS (Cell Therapy Systems) CD3/CD28 Dynabeads (Thermo Fisher Scientific, bead:cell ratio 1:1). After nucleofection, T cells were cultured in X-VIVO 15 media containing 500 U/ml IL-2 unless otherwise stated and split every two to four days. 

Cas9 RNP Electroporation
Electroporation was done as previously described (Roth et al., 2020). To produce ribonucleoproteins (RNPs), crRNA and tracrRNA (stock 160µM, both Dharmacon) were mixed 1:1 by volume, and annealed by incubation at 37C for 30 min to form an 80 µM guide RNA (gRNA) solution. Poly-L-glutamic acid (PGA, stock 125mg/ml, Sigma) was mixed with gRNA at 0.8:1 volume ratio prior to complexing with Cas9-NLS (QB3 Macrolab) for final volume ratio gRNA:PGA:Cas9 of 1:0.8:1 (Nguyen et al., 2020). These were incubated at 37°C for 15 min to form a 14.3 µM RNP solution. RNPs and HDRTs were mixed with T cells before electroporation (3.5 µl of RNP with 500ng/ µg=1µL of HDRT). Bulk T cells were resuspended in electroporation buffer P3 (Lonza Bioscience) at 0.75x 10^6 cells per 20 µl (per well) and transferred to a 96-well electroporation plate together with 4.5µL of RNP/HDRT mix per well. Pulse code EH-115 was used on a 4D-Nucleofector 96-well Unit (Lonza Bioscience). Cells were rescued in X-VIVO 15 without cytokines for 15 min and then cultured in X-VIVO 15 with 500 U/ml IL-2.