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

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