Culture: For pooled and arrayed Cas9 RNP screens and for GvHD experiments freshly isolated Treg cells were cultured in cRPMI with aCD3/CD28-coated beads (Stemcell technologies) in a 1:1 ratio. Starting day 2 of culture 300 -600 IU/ml IL-2 were added and replenished after 72 hrs and thereafter every 48 hrs 19. On day 9 of Treg cell expansion cells were restimulated for 48 hrs on plates coated overnight with 10 µg/ml aCD3 (clone: UCHT1; TONBO Biosciences or Biolegend) in cRPMI supplemented with 5 µg/ml aCD28 (clone: CD28.2; TONBO Biosciences or Biolegend).

Cas9 RNP assembly and electroporation: First, 80 µM crRNA (Dharmacon) and 80 µM tracrRNA (Dharmacon) were mixed in a 1:1 ratio and incubated for 30min at 37 °C to generate 40µM crRNA–trans-activating CRISPR RNA (tracrRNA) duplexes. An equal volume of 40 µM Streptococcus pyogenes Cas9-NLS (Macrolabs) was slowly added to the crRNA–tracrRNA and incubated for 15 min at 37 °C to generate 20µM Cas9 RNPs. For each editing reaction, 1.5-5x10^5 stimulated Treg cells were pelleted and re-suspended in 20 µL P3 buffer. 3 µl 20 µM Cas9 RNP and 0.75 µl 100 µM electroporation enhancer (IDT) was added directly to the cells and the entire volume transferred to a 96-well reaction cuvette (Lonza). Treg cells were electroporated using program EH-115 on the Amaxa 4D-Nucleofector (Lonza). 80 µL pre-warmed cRPMI was added to each well after electroporation and the cells were allowed to recover for 30 minutes at 37°C before restimulation.

Pooled Cas9 RNP screen: Treg cells were isolated, expanded and restimulated as described before. For the pooled Cas9 RNP-mix all 40 µM Cas9 RNPs were prepared separately as described before and mixed in equimolar ratios. 1.5x10^7 cells were suspended in 100 µl P3 electroporation buffer (Lonza) with 20 µl Cas9 RNP-mix and 6.6 µl 100 µM electroporation enhancer. Control cells were electroporated with equal amounts of non-targeting Cas9 RNP and electroporation enhancer (IDT). Treg cells were electroporated using program EH-115 on the 4D-Nucleofector (Lonza). 400 µl of pre-warmed cRPMI were added immediately after electroporation. After 30 min at 37°C cells were restimulated with 300 U/ml IL-2 and aCD3/aCD28-coated beads (Stemcell technologies) in a 1:1 ratio. After 48 hrs cells were split and stimulated with different cytokine combinations: 600 IU/ml IL-2 alone or 600 IU/ml IL-2 with 10 ng/ml IL-12 (Fisher Scientific), 10 ng/ml IL-4 (TONBO Biosciences), 10 ng/ml IL-6 (Fisher Scientific) and 10 ng/ml IFN-? (TONBO Biosciences) for 72 hrs.

Arrayed Cas9 RNP screen: Each of the 40 TFs was targeted with 3 different gRNAs (Dharmacon; predesigned crRNA library) in ex vivo expanded Treg cells to monitor potential off-target effects of individual gRNAs tested in the pooled Cas9 RNP screen...Electroporation of cells was performed using the P3 Primary Cell 96-well Nucleofector kit (Lonza) and 4D-Nucleofecter (Lonza; code: EH-115). Each reaction contained 2×10^5 expanded Treg cells, 3 µl of the respective Cas9 RNP and 0.75 µl of electroporation enhancer (IDT). After 48 hrs, cells were split and half were stimulated with 600 IU/ml IL-2 alone or 600 IU/ml IL-2 and 10 ng/ml IL-12 for 72 hrs.