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

Kyle Leix1, Candilianne Serrano-Zayas1, Hitarthi S Vyas1

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Researchers used CRISPR screening to identify 25 regions impacting LDLR gene expression, crucial for preventing heart attack and stroke. A key enhancer in the LDLR gene

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Area of Science:

  • Genetics
  • Molecular Biology
  • Cardiovascular Disease Research

Background:

  • Low-density lipoprotein receptor (LDLR) gene expression is vital for preventing atherosclerotic diseases like heart attack and stroke.
  • While sterol response elements are known regulators, other noncoding regions of the LDLR locus are poorly understood.

Purpose of the Study:

  • To functionally interrogate candidate cis-regulatory elements (CREs) at the LDLR locus using a high-throughput CRISPR screen.
  • To identify noncoding regions that significantly influence LDLR gene expression in its native genomic context.

Main Methods:

  • High-throughput CRISPR screening to assess the function of candidate LDLR CREs.
  • Reporter gene assays to validate enhancer activity.
  • Massively parallel reporter assays (MPRA) for fine-mapping enhancer function.

Main Results:

  • Identified 25 discrete genomic regions significantly impacting LDLR expression.
  • Validated an intronic region as a functional enhancer that reduces LDLR expression upon disruption.
  • Fine-mapped the enhancer to a conserved 129 bp interval containing transcription factor motifs and a GWAS-implicated variant (rs57217136).

Conclusions:

  • CRISPR screening is a powerful tool for dissecting the functional landscape of noncoding sequences.
  • Discovered novel CREs regulating LDLR, contributing to understanding atherosclerotic disease mechanisms.
  • Identified a specific enhancer linked to human LDL cholesterol levels, offering potential therapeutic targets.