Systematic discovery and perturbation of regulatory genes in human T cells reveals the architecture of immune networks [CRISPR]

Complex gene regulatory networks ensure that important genes are expressed at precise levels. When gene expression within these networks is sufficiently perturbed it can lead to disease. To understand how gene expression disruptions percolate through a network, we must first map the connections between regulatory genes and their downstream targets. However, it has been challenging to identify the upstream regulators for genes of interest. Here we develop a system for systematic discovery of upstream regulators of critical immune factors – IL2RA, IL-2, and CTLA4 – in primary human T cells. Then, CRISPR perturbations coupled with RNA-Seq, and ATAC-Seq, allowed us to map the full network of these regulators' target genes and enhancers. These regulators form highly interconnected networks with extensive feedback loops. Furthermore, this network is highly enriched for autoimmune-associated disease variants and genes. These results provide insight into how autoimmune associated disease genes are regulated in T cells as well as broader principles about the structure of human gene regulatory networks. Overall design: Pooled CRISPR screen followed by FACS to sort CD25 (IL2RA), IL-2, and CTLA4 high or low expressing cells.