XVivo15 medium (Lonza) supplemented with 5% fetal bovine serum, 50 mM 2-mercaptoethanol, and 10m MN-acetyl L-cystine was used to culture primary human T cells. In preparation for electroporation, T cells were stimulated for 2 days at a starting density of approximately 1 million cells per mL of media with anti-human CD3/CD28 Dynabeads (ThermoFisher), at a bead to cell ratio of 1:1, and cultured in XVivo15 media containing IL-2 (500 U/ml; UCSF Pharmacy), IL-7 (5 ng/ml; ThermoFisher), and IL-15 (5 ng/ml; Life Tech). Following electroporation, T cells were cultured in XVivo15 media containing IL-2 (500 U/ml) and maintained at approximately 1 million cells per mL of media. Every 2-3 days, electroporated T cells were topped up, with or without splitting, with additional media along with additional fresh IL-2 (final concentration of 500 U/ml). When necessary, T cells were transferred to larger culture vessels.

Primary T cell electroporation—For all electroporation experiments, primary T cells were prepared and cultured as described above. After stimulation for 48–56 hours, T cells were collected from their culture vessels and the anti-human CD3/anti-CD28 Dynabeads were magnetically separated from the T cells. Immediately before electroporation, debeaded cells were centrifuged for 10 min at 90g, aspirated, and resuspended in the Lonza electroporation buffer P3. Each experimental condition received a range of 750,000 – 1 million activated T cells resuspended in 20 µL of P3 buffer, and all electroporation experiments were carried out in 96 well format. All electroporations were done using a Lonza 4D 96-well electroporation system with pulse code EH115. Unless otherwise indicated, 3.5 µl RNPs (comprising 50 pmol of total RNP) were electroporated, along with 1–3 µl HDR Template at 1 µg/µl (1–3 µg HDR template total). Immediately after all electroporations, 80 µl of pre-warmed media (without cytokines) was added to each well, and cells were allowed to rest for 15 min at 37 °C in a cell culture incubator while remaining in the electroporation cuvettes. After 15 min, cells were moved to final culture vessels and media supplemented with 500 U/ml IL-2 was added.