Genetic and epigenetic screens in primary human T cells link candidate causal autoimmune variants to T cell networks [gRNA]

Genetic variants associated with autoimmune diseases are highly enriched within putative cis-regulatory regions of CD4+ T cells, suggesting that they alter disease risk via changes in gene regulation. However, very few genetic variants have been shown to affect T cell gene expression or function. Here, we tested >18,000 autoimmune disease-associated variants for allele-specific expression using massively parallel reporter assays in primary human CD4+ T cells. We find 545 variants that modulate expression in an allele-specific manner (emVars). Primary T cell emVars greatly enrich for probable causal variants, are mediated by common upstream pathways, and their putative target genes are highly enriched within a lymphocyte activation network. Using bulk and single-cell CRISPR-interference screens, we confirm that emVar-containing T cell cis-regulatory elements modulate both known and previously unappreciated target genes that regulate T cell proliferation, providing plausible mechanisms by which these variants alter autoimmune disease risk. We created gRNA libraries targeting on average 14 gRNAs to each variant CRE (within 100 bp of each variant), 120 positive control gRNAs targeting known regulators of T cell proliferation and effector function, and 992 non-targeting control gRNAs We activated primary human T cells and delivered dCas9- ZIM3 and the gRNA library using lentivirus infection. We harvested half of the transduced cells directly post-puromycin treatment (day 2) and the remainder of the cells after 21 days of proliferation (day 21).