Functional CRISPR dissection of gene networks controlling human regulatory T cell identity

Schumann K, Raju SS, Lauber M, Kolb S, Shifrut E, Cortez JT, Skartsis N, Nguyen VQ, Woo JM, Roth TL, Yu R, Nguyen MLT, Simeonov DR, Nguyen DN, Targ S, Gate RE, Tang Q, Bluestone JA, Spitzer MH, Ye CJ, Marson A
Source: Nat Immunol
Publication Date: ()
Issue: 11: 1456-1466
Research Area:
Immunotherapy / Hematology
Cells used in publication:
T cell, human stim.
Species: human
Tissue Origin: blood
4D-Nucleofector® 96-well Systems

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.


Human regulatory T (Treg) cells are essential for immune homeostasis. The transcription factor FOXP3 maintains Treg cell identity, yet the complete set of key transcription factors that control Treg cell gene expression remains unknown. Here, we used pooled and arrayed Cas9 ribonucleoprotein screens to identify transcription factors that regulate critical proteins in primary human Treg cells under basal and proinflammatory conditions. We then generated 54,424 single-cell transcriptomes from Treg cells subjected to genetic perturbations and cytokine stimulation, which revealed distinct gene networks individually regulated by FOXP3 and PRDM1, in addition to a network coregulated by FOXO1 and IRF4. We also discovered that HIVEP2, to our knowledge not previously implicated in Treg cell function, coregulates another gene network with SATB1 and is important for Treg cell-mediated immunosuppression. By integrating CRISPR screens and single-cell RNA-sequencing profiling, we have uncovered transcriptional regulators and downstream gene networks in human Treg cells that could be targeted for immunotherapies.