For electroporation of 1 × 10^6 cells, 20 µL of resuspended cells were transferred to 1.88 µL of RNP/HDRT (except during DNA-escalation studies where different volumes were used) and mixed thoroughly. Afterward, the T cell/RNP/HDRT mixture was transferred onto a 16-well electroporation strip (20 µL = 106 cells per well, Lonza) or an electroporation cartridge (100 µL = 5 × 10^6 cells, Lonza). Cells were carefully transferred onto the electroporation strips using 200-µL tips to avoid trapping air in the solution. Electroporation strips and cartridges were tapped onto the bench several times to ensure correct placement of fluids within the electroporation vessel. Electroporation was performed on a 4D-Nucleofector Device (Lonza) using the program EH-115. Directly after electroporation, pre-warmed T cell medium was added onto the cells (90 µL per well and 450 µL per cartridge). Afterward, resuspended cells were transferred to 96-well round-bottom plates (50 µL/well) containing 150 µL pre-warmed T cell medium per well (with or without HDR-enhancing supplements) at a density of 0.5 × 10^6 cells per well.

For large-scale CAR T cell generation by electroporation in cartridges, 950 µL of pre-warmed T cell medium was used for initial resuspension. Ten minutes after electroporation, T cells were transferred into 24-well flat-bottom plates (500 µL/well) containing 1.5 mL of T cell medium (with or without HDR-enhancing supplements) at a density of 2.5 × 10^6 cells per well.

Jurkat cells (Clone E6-1, ATCC TIB-152) were cultured in RPMI media (ATCC) supplemented with 10% fetal bovine serum (FBS) (Gibco) and 1% Pen Strep (Gibco), maintained at a density between 10^5 and 10^6 cells/mL. Cas9 gRNA targeting HPRT1 was prepared by mixing equimolar amounts of Alt-R CRISPR-RNA (crRNA) and Alt-R trans-activating CRISPR RNA (tracrRNA) (IDT, Coralville, IA, USA) in Tris-EDTA (TE), heating to 95°C, and slow cooling at room temperature. A single-stranded DNA oligo consisting of 40 nt of homology flanking the Cas9 cleavage site and a 6-base EcoRI (5'-GAATTC-3') restriction site inserting at the Cas9 cleavage site was designed (Table S3); the non-targeting (guide-containing) sequence strand was ordered as an Alt-R HDR Donor Oligo (IDT). The RNP was formed by complexation of 56-pmol IDT Cas9 protein with 67.2-pmol gRNA complex in a total volume of 7.5 µL with PBS to adjust to the final volume. All reagents were delivered to Jurkat cells using the Lonza Nucleofector System. Cells were counted and pelleted by centrifugation (300g, 10 min at room temperature) and washed with 10 mL 1× PBS. The cells were again pelleted and resuspended in Lonza Nucleofection Solution SE at a density of 1 × 10^8 cells/mL. For each electroporation, 5 µL of RNP was added to 20 µL of Jurkat cells in Nucleofection Solution SE (5 × 10^5 cells/nucleofection). Donor DNA and IDT Alt-R Cas9 Electroporation Enhancer were added to achieve a final concentration of 1 and 3 µM, respectively, in a total reaction volume of 28 µL. The solution was mixed by pipetting, and 25 µL was transferred to an electroporation cuvette plate. The cells were electroporated according to the manufacturer’s protocol using the Lonza 96-well Shuttle and nucleofection protocol 96-CL-120.