PROJECT SUMMARY Dilated cardiomyopathy (DCM) is a leading cause of morbidity and mortality worldwide, with an estimated prevalence of 1:250 in the general population. Recent human genetic studies suggest that the complex heritability of DCM may be due to a combination of polygenic causes. However, the complex interactions among various genetic variants are less understood, hampering the development of effective therapeutics for DCM. To achieve the latter, we will use state-of-the-art approaches by employing the induced pluripotent stem cell-derived cardiomyocyte (iPSC-CM) platform coupled with tissue engineering, single cell RNA sequencing (scRNA-seq), and CRISPR/dCas9 genome screening technologies to interrogate the interplay between genetic variants in the etiology of DCM and identify novel drug target for polygenic DCM treatment. In Aim 1, we will generate iPSC lines from 20 polygenic DCM patients and 10 controls from the same family cohort, and create 120 genome- edited isogenic iPSC lines using CRISPR/Cas9. We will then differentiate the polygenic DCM iPSCs and corresponding isogenic iPSC lines to iPSC-CMs. Using bioinformatic analysis, we will generate a gene expression “score index” to assess the pathogenicity of each genetic variant and interactions between different genetic variants. In Aim 2, we will generate 3D engineered heart tissues (EHTs) to elucidate functional consequences of polygenic DCM variants at the 3D level. We will make 3D EHTs consisting of iPSC-CMs, iPSC- derived endothelial cells (iPSC-ECs), and iPSC-derived fibroblasts (iPSC-FBs) from these 20 polygenic DCM patients and their genome-edited isogenic iPSC lines, which allow us to simulate complex cell-cell interactions and understand the crosstalk among different cell types. In Aim 3, we will use CRISPR/dCas9 genome screening approach in combination with scRNA-seq technology to identify genes suitable as drug targets in polygenic DCM iPSC-CMs. This novel technique provides a unique and cost-efficient way to systemically screen for druggable genes associated with polygenic DCM. Collectively, the proposed aims will help us comprehensively elucidate the genetic and molecular basis of polygenic DCM and discover novel drug targets for patient-specific therapeutics.