Functional interrogation of candidate cis-regulatory elements at the LDLR locus

Regulation of LDLR gene expression plays an important role in the development of atherosclerotic diseases including heart attack and stroke. Although LDLR regulation by sterol response elements has been well characterized, the functional significance of other noncoding regions at the LDLR locus remains poorly defined. We developed and applied a high throughput CRISPR screen to test the functional significance of candidate LDLR cis-regulatory elements in their native genomic context. Analysis of our screen results revealed a discrete region in the first intron of LDLR with a significant impact on cellular LDL uptake. We validated the presence of enhancer activity in this region by confirming that its disruption reduced endogenous LDLR expression while its insertion upstream of a minimal promoter augmented reporter gene expression. We then applied a massively parallel reporter assay to fine map enhancer activity in this region to a 129 bp interval that is highly conserved among vertebrates, exhibits biochemical hallmarks of enhancer activity, is enriched for transcription factor binding motifs, and contains a common genetic variant (rs57217136) that has been associated with human LDL cholesterol levels by genome-wide association studies. Overall, these findings demonstrate the power of CRISPR screening to interrogate candidate CREs and support the functional significance of an enhancer in the first intron of LDLR.