Tregs were cultured in X-VIVO 15 (Lonza) with 5% human serum from male AB plasma (Sigma-Aldrich), IL-2 (300 U/ml; PeproTech), and 100 nmol rapamycin (STEMCELL Technologies; only added for certain experiments). Teff cells were cultured in X-VIVO 15, 5% human serum, and IL-2 (50 U/ml). The Tregs and Teff cells were cultured at 37°C with 5% CO2 and ambient oxygen levels. Treg-like MT-2 cells were cultured in X-VIVO 15 with 5% human serum and 1% penicillin/streptomycin. K562 cells (American Type Culture Collection) were cultured in RPMI 1640 medium (Thermo Fisher Scientific) with 10% fetal bovine serum (Thermo Fisher Scientific) at 37°C with 5% CO2 and ambient oxygen levels. For all cells, fresh medium was added every 2 to 3 days.

For the initial screen, 2µg of sgRNA/Cas9 plasmid DNA was nucleofected into 1 million K562 cells using the Lonza Nucleofector 2b (program T-016).

The sgRNA was complexed with Cas9 for 10 min at 25°C at an approximate Cas9:sgRNA molar ratio of 1:2.5, using 8 µg of sgRNA and 15 µg of Cas9 per 100 µl of nucleofection solution containing 2.5 × 105 to 1 × 106 cells. After switching to high-fidelity (HiFi) Cas9 (IDT) that showed slightly lower efficiency in parallel experiments, the amount of HiFi Cas9 was increased, and the molar ratio was adjusted to 1:1.8, using 8 µg of sgRNA with 22 µg of HiFi Cas9 per 100 µl of nucleofection solution. The sgRNA/Cas9 complexes (IDT, TriLink BioTechnologies and Synthego) were nucleofected into Tregs and Teff cells after 2 to 3 days of activation using the P3 Primary Cell Nucleofection Kit (Lonza) and the Lonza Nucleofector 4D (program E-0115). For HSPC editing, the cells were nucleofected using the P3 Primary Cell Nucleofection Kit (Lonza) and the Lonza Nucleofector 4D (program DZ-100).