Cultivation: 4 biological replicates of sorted adult or fetal naïve T cells were stimulated in U-bottomed 96 wells with 5 µl of Immunocult Human T cell Activator (CD3/CD28/CD2 tetramers, STEMCELL Technologies) in 200 µl of culture media (RPMI-1640 supplemented with 10% HI-FBS (heat inactivated fetal bovine serum), 300 mg/L L-glutamine, 10 U/mL penicillin, and 10 µg/mL streptomycin, 10 mM HEPES, 1X MEM-NEAA, ß-mercaptoethanol). Exogenous IL-2 (10 ng/ml, Peprotech) and TGF-ß (50 ng/ml, Peprotech) were added according to the relevant experimental setup.
CRISPR-Cas9 editing of the Helios locus:
Cas9 gRNA ribonucleoproteins (RNPs) were assembled before ex-periments by assembling the gRNAs through incubating 160 µM CRISPR RNA (crRNA) with 160 µM trans-activating crRNA (tracrRNA) (Dharmacon) at a 1:1 ratio for 30 min at 37°C to a final concentration of 80 µM.Assembled gRNAs were then subsequently incubated with 40 µM Cas9-nuclear localization sequence (NLS) (QB3 MacroLab, University of California, Berkeley) at a 1:1 ratio for 15 min at 37°C for a final concentration of 20 µM RNPs. Nucleo-fection was performed using the Amaxa P3 Primary Cell 96-well Nucleofector Kit and 4D-Nucleofector (Lonza). A total of 1 × 10^5 sorted fetal naïve T cells per well were stimulated overnight in 96-well plates precoated with anti-CD3 antibodies (1 µg/ml; HIT3a, BD Biosciences) and supplemented with soluble anti-CD28 antibodies (2 µg/ml; CD28.2, BD Biosciences) and IL-2 (10 ng/ml; PeproTech). Stimulated fetal naïve T cells were washed with PBS and resuspended in 20 µl of P3 solution. Five microliters of the final 20 µM RNP solution was added, along with 1 µl of 100 µM homology directed repair template (HDRT) solution. Cells were gently mixed and transferred to the 96-well shuttle device. Cells were electroporated using program EH-115 on the Amaxa 4D-Nucleofector (Lonza).