Multimodal CRISPR perturbations of GWAS loci associated with coronary artery disease in vascular endothelial cells

Author summaryGenome-wide association study (GWAS) is a method designed to identify genetic variants that increase the risk to develop common human diseases such as heart attacks or schizophrenia. While powerful, this method has one major limitation: it cannot unambiguously pinpoint the genes responsible for the diseases. This important step requires investigators to experimentally test genes located near the variants identified by GWAS for functions related to the diseases of interest. Here, we used a technique called CRISPR to test if genes near genetic variants implicated by GWAS in heart attacks modulate the functions of vascular endothelial cells. Endothelial cells form the inner layer of blood vessels, and play a critical role in the development of the pathology (atherosclerosis) that leads to heart attacks. In total, we found 26 regions in the human genome that include heart attacks-associated variants and that influence the functions of endothelial cells. Focusing specifically on one of our findings, we showed that the gene DHX38 regulates a process called senescence, which in turn modulates how endothelial cells respond to stimuli that promote heart attacks. Genome-wide association studies have identified >250 genetic variants associated with coronary artery disease (CAD), but the causal variants, genes and molecular mechanisms remain unknown at most loci. We performed pooled CRISPR screens to test the impact of sequences at or near CAD-associated genetic variants on vascular endothelial cell functions. Using CRISPR knockout, inhibition and activation, we targeted 1998 variants at 83 CAD loci to assess their effect on three adhesion proteins (E-selectin, ICAM1, VCAM1) and three key endothelial functions (nitric oxide and reactive oxygen species production, calcium signalling). At a false discovery rate <= 10%, we identified significant CRISPR perturbations near 42 variants located within 26 CAD loci. We used base editing to validate a putative causal variant in the promoter of the FES gene. Although a few of the loci include genes previously characterized in endothelial cells (e.g. AIDA, ARHGEF26, ADAMTS7), most are implicated in endothelial dysfunction for the first time. Detailed characterization of one of these new loci implicated the RNA helicase DHX38 in vascular endothelial cell senescence. While promising, our results also highlighted several limitations in using CRISPR perturbations to functionally dissect GWAS loci, including an unknown false negative rate and potential off-target effects.