Abstract 1024: A Genome-wide CRISPR Screen Identifies The LDL Receptor As The Primary Mediator Of Lp(a) Uptake By HuH7 Cells

Abstract

Elevated Lp(a) blood levels have been causally linked to the development of atherosclerotic and valvular heart disease, yet the identity of the specific receptor(s) that mediate hepatic clearance of Lp(a) remains controversial. Here, we report the development, execution, and validation of a genome-wide CRISPR screen for functional modifiers of Lp(a) uptake by HuH7 cells. We first optimized a method for fluorescently labeling purified human Lp(a). Sensitive and specific detection of fluorescent Lp(a) uptake by HuH7 cells was performed by flow cytometry. We then used this approach to isolate subpopulations of individual cells with aberrant Lp(a) uptake from a pool edited with a library of 123,411 gRNAs targeting 19,050 genes across the human genome. Strikingly, the top positive regulator of Lp(a) uptake in our screen was LDLR , which encodes the low-density lipoprotein receptor. MYLIP , which encodes a ubiquitin ligase that degrades LDLR, was the top negative regulator. We also observed a significant influence for several other established LDLR regulators, including those with a canonical role in SREBP signaling and those discovered in our analogous prior screen of LDL uptake. No other previously proposed Lp(a) receptor had a significant contribution to Lp(a) uptake in our screen. We subsequently validated our screen results by engineering HuH7 cell lines with targeted deletion or overexpression of LDLR and confirmed their corresponding impact on Lp(a) uptake. Lastly, we used biolayer interferometry to detect in vitro binding between purified Lp(a) and recombinant LDLR ectodomain. Together, our findings are consistent with a model in which hepatic clearance of Lp(a) is primarily mediated by the LDL receptor.