ABSTRACT Systematic lupus erythematosus (SLE or lupus) is a currently incurable autoimmune disease, characterized by abnormal immune cell (e.g., B-lymphocytes) response and the production of numerous pathogenic autoantibodies, culminating in multi-organ damage (e.g., kidneys, skin). While the etiology of SLE is incompletely understood, a substantial genetic contribution is well established. Several genome-wide association studies (GWAS) have identified over 100 SLE predisposing loci (p<5x10-8), mostly single nucleotide polymorphisms (SNPs). Most of these SNPs do not directly alter protein products, and previous work from us and others have shown that many such SNPs are enriched within cis regulatory elements (cRE) (i.e., enhancers and silencers) and likely to modulate gene expression. However, pinpointing the causal, predisposing SNPs within cREs and deciphering their precise mechanisms represent major obstacles to progress in the field. Consequently, this knowledge gap has severely hindered the translation of GWAS findings into clinical applications. Hence, there is a profound need for unbiased, comprehensive, and high-throughput approaches to address the mechanistic link between hundreds of potential regulatory SNPs (rSNPs) and SLE susceptibility. We hypothesize that SLE- predisposing rSNPs aberrantly induce cRE activities that influence the expression of target genes in unstimulated and/or stimulated B-cells. To systematically delineate rSNPs and their impact on target genes, we propose to establish a high-throughput experimental pipeline to determine and validate rSNPs underlying GWAS loci. In Aim 1, we will apply the high-throughput technique “Self-Transcribing Active Regulatory Region-sequencing” (STARR-seq) to functionally quantify the regulatory activities of hundreds of SNP-containing regions simultaneously. Using Raji cells (B-lymphocyte) in both unstimulated and stimulated conditions, we will apply STARR-seq to evaluate 756 selected rSNPs within 79 distinct GWAS loci for SLE susceptibility. In Aim 2a, we will apply next-generation (NG)-Capture-C to detect SNP-specific effects on cis interactions with endogenous, cognate target genes in Raji cells, needing no strong a priori hypothesis of target genes and functional consequences. We will evaluate the same set of 756 SNPs in Aim 1 and Aim 2a. We anticipate this two-prong tandem strategy will bridge the gap between GWAS-derived rSNPs and mechanistic links to their target genes. To validate the effectiveness and accuracy of the proposed methods, Aim 2b will use CRISPR-based (epi)genetic editing of a selected rSNP to validate the allele-specific functional effects on the endogenous target gene(s) in isogenic cells. Collectively, the proposed unbiased approaches for discovering and validating SLE “causal” SNPs is high risk/high reward and may lead to breakthroughs in the understanding of SLE etiology and intervention strategies. Discovery of rSNPs, cREs, and their target genes will significa...