Gene editing
Primary human T cells were harvested ~48 h after anti-CD3/CD28 stimulation, counted, and washed twice in sterile PBS by centrifugation at 100 ×g for 10 min at RT.
mRNA-based knockin
Double-nick-mediated knockins were performed by co-electroporating: (1) HDR donor template (1 µL, 1–2 µg/µL), (2) two strand-specific sgRNAs (0.48 µL each, 100 µM; IDT), and (3) mRNA encoding the base or nuclease editor (1 µL, 2 µg/µL). Optionally, additional sgRNAs for base editing were included. Reagents were assembled in PCR strips by adding components sequentially. Synthetic, chemically modified sgRNAs (or crRNAs for Cas12a; IDT) were resuspended in nuclease-free 1×TE buffer and stored at 20°C (Table S2). sgRNAs for splice site disruption using base editors were designed using SpliceR.29Previously published sgRNAs targeting TRAC, B2M,40CIITA,40CD54, CD58, CD38, CD2,73CD5,74and CD774 were used.
Cas12a ribonucleoprotein-based knockin
For Cas12a RNP assembly, 0.5 µL of 100 µg/µL poly(L-glutamic acid) (PGA; 15–50 kDa; Sigma-Aldrich) was combined with 0.48 µL TRAC-specific CRISPR-RNA (crRNA) (IDT) and mixed thoroughly. Then, 0.4 µL of Alt-R A.s. Cas12a Ultra (IDT; 10 µg/µL) was added and incubated for 15 min at RT to form RNPs, followed by addition of 1 µL HDRT (1–2 µg/µL). The mixture was kept on ice until electroporation.
Electroporation
Cells were resuspended in 20 µL of ice-cold P3 electroporation buffer (Lonza) at a concentration of 1–1.5 ×10^6 cells per reaction and immediately used for electroporation. The cell suspensions were mixed with the prepared reagent mix and transferred to a 16-well electroporation strip (Lonza). To ensure proper contact and eliminate air bubbles, strips were gently tapped on the bench before electroporation.
Electroporation was performed on a 4D-Nucleofector (Lonza) using program EH-115. Immediately after, 100 µL of pre-warmed T cell medium was added per well and the cells were placed in the incubator for 10 min. Cells were then gently resuspended and equally divided into two separate wells of a 96-well round-bottom plate, each containing 150 µL of pre-warmed T cell medium, resulting in a final density of 0.5– 0.75 ×10^6 cells per well. For the animal experiment, three electroporation reactions were performed and subsequently seeded together in one well of a G-Rex24 Well Plate (3 M cells per well).