Cas9 RNP assembly and electroporation: 80 µM crRNA (Dharmacon) and 80 µM tracrRNA (Dharmacon) were mixed in a 1:1 ratio and incubated 30 min at 37°C to generate 40 µM crRNA:tracrRNA duplexes. An equal volume of 40 µM S. pyogenes Cas9-NLS (Macrolabs, Berkeley) was slowly added to the crRNA:tracrRNA and incubated for 15 min at 37°C to generate 20 µM 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: For the pooled Cas9 RNP-mix all 40 µM Cas9 RNPs were prepared separately as described before and mixed in equimolar ratios. 1.5x107 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: 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.