Abstract

Drug targets with human genetic evidence are expected to increase clinical success by at least two-fold. Yet, translating disease-associated genetic variants into functional knowledge remains a fundamental challenge of early drug discovery. To aid the identification and characterisation of genetically supported targets, we developed a single cell CRISPR based, variant-to-gene-to-function (V2G2F) pipeline in primary T cells. A key gap/opportunity in the V2G space is that currently only ~15% of immune disease associations can be “cleanly” mapped to a gene. Immune disease associated variants are enriched within regulatory elements, such as distal enhancers, found in cell type-specific open chromatin regions. To enable the mapping of high interest immune disease associated elements to genes at scale, we established a “crisprQTL” pipeline in primary human T cells. We used this framework to screen 244 distal, immune disease-associated regulatory elements in T cells (derived from 4 donors) and identified 76 novel element-to-gene links. Next, we sought to elucidate the function of these gene hits (i.e. G2F mapping) in the context of CD4 T cell differentiation. Gene knock-out CRISPR screens leveraging SLICE technology (lenti sgRNA + Cas9 RNP) in primary T cells have been previously reported in the literature. We developed “SLICE&Skew CROP-CITEseq” by coupling targeted gene knock-outs in primary T cells differentiated in specific cytokine cocktails with single cell RNAseq. This framework allowed us to query over 1000 genes, dissect the molecular mechanism underlying gene function and prioritise novel targets for drug development. In conclusion, we present a functional genomics approach that enables the generation of enhancer-gene-function maps at scale in primary T cell differentiation models, opening up new avenues for genetics and functional genomics driven target identification.