PROJECT SUMMARY/ABSTRACT I propose to develop a novel assay, single-cell Massively Parallel Reporter Assay (scMPRA), to study gene by environment interactions (GxE) and in particular gain a greater understanding of how nucleotide variation in cis-regulatory elements interact with the variable environments of single cells to modulate gene expression. I hypothesize that the cellular environment modifies gene expression by modulating the activity of regulatory elements and these can behave differently due to nucleotide variants. Currently, there are no high-throughput methods available to functionally validate variants across different cellular environments. To address this, I will adapt a lentivirus based Massively Parallel Reporter Assay (lentiMPRA) developed in our lab and use it in combination with 10x Genomics scRNA-seq technology to allow the simultaneous quantification of the cellular environment via the transcriptome and of enhancer activity with the lentiMPRA construct. This assay will provide a novel platform to test the causality of variants driving changes in gene expression in specific environments in a high throughput manner. I will use this technology to study and validate GxE in distinct cell types (Aim1) and in different immune stimulation conditions (Aim 2) to dissect the relationship between immune activation and underlying genetic variation. We will use linear mixed models (LMMs) to quantify the interaction between SNPs and environments including, cell type, cell state, and donor. This work will map enhancers and their disease associated variants that are sensitive to the cellular environment, providing a platform to assign causality to variants that interact with specific environments.