Regulators of Type III Cytokines in Type III Innate lymphoid Cells Identified by CRISPR Activation and Inhibition Screens

Abstract Type III cytokines interleukin (IL)-22 and IL-17 are expressed by both innate and adaptive immune cells to defend against extracellular pathogens, yet their dysregulation contributes to autoimmunity and malignancy. A deeper knowledge of IL-22/17 regulation is warranted to understand physiologic processes as well as disease pathogenesis, thereby elucidating druggable targets. Toward this goal, we applied genome-wide CRISPR activation (CRISPRa) and CRISPR inhibition (CRISPRi) screens to identify the collection of factors that positively or negatively regulate IL-17/22 expression in a murine type III innate lymphoid cell (ILC3) model. Following lentiviral transduction of a CRISPRi or CRISPRa guide RNA (gRNA) library, cells were stimulated and sorted by levels of intracellular IL-22/17 protein expression. Relative gRNA frequencies in each sequenced population revealed enrichment and depletion of potential regulators (“hits”), which were then tested with gene-specific gRNAs to validate or exclude them from further study. These genome-wide screens have illuminated hits from several biological pathways, ranging from transmembrane channels to RNA splicing. We identify previously known regulators, including IL23R, STAT3, and ZFP36, as well as novel potential regulators, including SLC family members and a nuclear protein called SON. Our screens provide a diverse array of candidate factors that govern expression of type III cytokines by ILC3s, with potential for extrapolation to other cytokine-expressing cells. Genome-wide gain and loss of function screens are powerful, complementary tools to facilitate regulatory pathway discovery.