Kyle Leix, Candilianne Serrano-Zayas, Hitarthi S. Vyas, Sarah E. Graham, Brian T. Emmer
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. In this study, we developed and applied a high throughput CRISPR screen to test the functional importance of candidate LDLR cis-regulatory elements (CREs) in their native genomic context. In total, we found 25 discrete regions to exhibit a significant impact on LDLR expression. For one of these regions with particularly strong activity in the first intron, we validated the presence of an enhancer 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 within 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 clarify the functional landscape of noncoding sequences at the LDLR locus.
Kyle Leix, Candilianne Serrano-Zayas, Hitarthi S. Vyas, Sarah E. Graham, Brian T. Emmer. Functional interrogation of candidate cis-regulatory elements at the LDLR locus. 2026-03-17. https://doi.org/10.1371/journal.pgen.1012082
A lentiviral MPRA library tested 74 overlapping tiles spanning an approximately 860 bp candidate enhancer interval in the first intron of human LDLR, together with three positive and three negative control sequences. HuH7 cells received five independent library transductions, and reporter activity was measured two days later as barcode cDNA abundance relative to genomic DNA.
A lentiviral MPRA library tested 74 overlapping tiles spanning an approximately 860 bp candidate enhancer interval in the first intron of human LDLR, together with three positive and three negative control sequences. HepG2 cells received five independent library transductions, and reporter activity was measured two days later as barcode cDNA abundance relative to genomic DNA.
Original supplemental and deposited inputs retained for this study. Download files individually or together as a ZIP; nested folders are preserved. Source reuse terms apply, and sequencing reads may be omitted.
Download all 7 files (ZIP)S10_oligonucleotides.xlsxS5_MPRA_library_synthesis_QC.tifS6_MPRA_library_design.xlsxS6_MPRA_reproducibility_QC.tifS7_barcode_CRS_associations.xlsxS8_MPRA_expression_counts.xlsxS9_MPRA_expression_analysis.xlsx