Treg Stimulation
After isolation, Tregs were cultured for six days in 24-well plates at 250.000 cells/well in 1 mL X-vivo (BE02-060F, Lonza) + 5% heat-inactivated fetal bovine serum (FBS) (S1400, Biowest) with 10 µg/mL plate-bound anti-CD3 (555329, BD Biosciences), 1 µg/ mL soluble anti-CD28 (555725, BD Biosciences) and 300 U/mL IL-2 (11147528001, Sigma-Aldrich) or 1500 IU/mL Proleukin® (Novartis). For short term expansion experiments, Tregs were cultured for 24 hours in above-mentioned conditions and underwent subsequent nucleofection without prior re-plating to 6-well plates.

Treg Nucleofection
24 hours prior to nucleofection, cells were cultured in 6-well plates at a density of 250.000 cells/mL in 2 mL X-vivo + 5% FBS and 100 U/mL IL-2 (11147528001, Sigma-Aldrich) or 500 IU/mL Proleukin® (Novartis). For transfection, cells were collected, centrifuged at 90g for 10 minutes at room temperature, and 1 million Tregs were resuspended in 20 µl P3 Primary Cell 4D-Nucleofector X Kit S (V4XP-3032, Lonza). In PCR tubes, 20 pmol Cas9 nuclease (9212-0.25MG, Aldevron) was mixed with
100 pmol sgRNA (Synthego, Table S1) and incubated at 37°C for a minimum of 10 minutes before adding to the cells. For multiplexing, RNP complexes for each sgRNA were generated separately and equal amounts of each sgRNA was added. The cell/RNP mixture was transferred to Nucleofection cuvette strips (4D-Nucleofector X Kit S, Lonza) and cells were electroporated using the 4D-Nucleofector Core Unit (AAF-1002B, Lonza) and X Unit (AAF-1002X, Lonza) with program EO115. After transfection, 80 µl medium at room temperature (X-vivo + 5% FBS + 100 U/mL IL-2 or 500 IU/mL Proleukin®) was added to the wells of the cuvette strip. Cells were collected and plated in 1 mL pre-warmed medium in 24-well plates and incubated at 37° C until read-out. For re-stimulation, cells were activated 2 hours to 4 days after nucleofection with anti-CD3 plate bound mAb (1 – 10 µg/mL) and 1 µg/mL soluble anti-CD28 (555725, BD Biosciences) in the presence of IL-2.