Abstract Body: The expression level of the Low-Density Lipoprotein Receptor (LDLR) gene is a major determinant of one’s lifetime risk for atherogenic cardiovascular diseases (ASCVDs). LDLR encodes a cell surface protein that mediates the clearance of low-density lipoprotein (LDL) from the bloodstream. Dysregulation of LDLR expression is linked to the development of ASCVDs, which affect approximately 1 in 15 people worldwide. While much is known about the mechanisms regulating LDLR expression, the role of non-coding cis-regulatory elements remains poorly understood. Here, we aim to define the functional landscape of the LDLR locus and to translate this knowledge into a novel genome editing approach for ASCVD treatment.
We are performing a high-throughput CRISPR screen employing three approaches: CRISPR activation, CRISPR inhibition, and classical CRISPR-mediated indel formation. Each method utilizes a uniquely modified Cas enzyme to drive activation, inhibition, or indel formation, respectively. To validate these systems, we primarily performed a pilot study using a set of single-guide RNAs (sgRNAs) targeting non-coding regions of LDLR, along with a pre-validated positive control targeting the coding region. Our results confirmed that three of four modified Cas systems— VP64, KRAB_dCas9, and Lenti-Cas9 BLAST—were functional, whereas pXPR_120 did not show great activation response. This success led us to proceed with a large-scale screen to fully map the functional landscape of the LDLR locus. For the screen, we have developed a library of 16,402 sgRNAs targeting regions within 98 kb of the LDLR locus. The library, constructed in lentiGuide-Puro and pXPR_502 backbones, was validated through next-generation sequencing (NGS), achieving 99.5% coverage.
In conclusion, the validated CRISPR strategies are functional and arranged for large-scale
screen that, when put together, shall develop a detailed functional map of the LDLR locus, aiding the development of advanced and novel genome editing therapeutic options for ASCVDs.