Abstract 117: A Genome-wide CRISPR/Cas9 Knockout Screen Reveals New Targets To Increase Cellular LDL Uptake.

Identifying new genetic factors involved in lipoprotein metabolism will provide new targets to lower plasma lipids and prevent cardiovascular disease; however, genetic epidemiological studies failed to recognize all the contributors to plasma lipids levels. Recently, using a CRISPR-Cas9 knockout screen in HepG2 cells, we found that knocking out transgelin, an actin-binding protein, reduces LDL uptake in hepatoma cells by interfering with the actin-dependent phase of the clathrin-mediated endocytosis (CME). An ideal therapeutic target to reduce plasma LDL would be one like PCSK9 in which, once knocked out or inhibited, cellular LDL uptake would increase. Aim: To identify genetic factors that, by knocking them out, increase LDL uptake in hepatoma cells. Methods and Results: We performed a whole-genome CRISPR-Cas9 knockout screen (targeting 19,144 genes) on HepG2 cells, sorting for cells with increased LDL uptake. Gene enrichment was analyzed with MAGeCK software. As expected, MYLIP , a mediator of LDL receptor ubiquitination and degradation, was among the most enriched genes ( ranked #9, log fold change: 4.82 ). The 200 top-enriched genes (1%) were analyzed for associations with plasma lipid traits using the NHLBI-GRASP (v2.0.0.0). 35 out of 200 genes had associations with p<2.5x10-4 (0.05/200) and were considered for further in vitro analysis. Stable knockout HepG2 cells were generated for a subset of these candidate genes and were tested for LDL uptake. Increased in LDL uptake was observed for: MYLIP , +66%; HUNK , +38%; PACSIN1 , +41%; PACSIN3 , +55%; RAI14 , +75%; PSPH , +134% (% to control, p<0.01 for all). Interestingly, these genes showed interactions with CME-related genes in a protein-protein interactome analysis, revealing interactions with genes related to clathrin-coated pit and actin filament dynamics. Conclusion: We found novel genetic factors associated with LDL internalization by applying a validated whole-genome gene editing tool. Our data suggest that these factors may be involved in different steps of LDL endocytosis and may function as possible therapeutic targets to increase hepatic LDL clearance. Further studies will be needed to elucidate the mechanisms underlying these associations.