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 FOXP3 maintains T_reg cell identity, yet the complete set of key transcription factors that control T_reg 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 T_reg cells under basal and proinflammatory 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 coregulated by FOXO1 and IRF4. We also discovered that HIVEP2, to our knowledge not previously implicated in T_reg cell function, coregulates another gene network with SATB1 and is important for T_reg cell-mediated immunosuppression. By integrating CRISPR screens and single-cell RNA-sequencing profiling, we have uncovered transcriptional regulators and downstream gene networks in human T_reg cells that could be targeted for immunotherapies.