CRISPR/Cas9 Whole-Genome Screens Identify Genes Critical for Neutral Lipid Metabolism in Macrophages

Lipid-laden macrophages contribute to atherosclerotic plaque formation in atherosclerosisrelated heart diseases. Thus, understanding formation and degradation of lipid would help identify therapeutic targets for the treatment of atherosclerosis. In the first chapter of this dissertation, I have explained the current understanding of cholesterol metabolism in macrophages and how mTOR could play a potential role in this process. Additionally, I have described how CRISPR/Cas9 whole-genome screens present an effective tool to identify genes regulating cholesterol metabolism including positive and negative regulators of lipid droplet formation and lipid droplet degradation. In Chapter 2, I have described the results of the CRISPR/Cas9 whole-genome screens that identified critical genes regulating macrophage cholesterol metabolism processes. Specifically, we have identified essential genes and their potential pathways involved in cholesterol metabolism. Using CRISPR/Cas9 targeted gene disruption, we successfully recapitulated screen phenotype suggesting the validity of screen hits. This work opens the door for performing hypothesisdriven studies to understand the biology of cholesterol metabolism in macrophages. In Chapter 3, I have described the results of our research in investigating the role of mTOR regulating genes in lipid droplet degradation. We have observed that mTOR regulating genes mediate lipid droplet degradation via positive regulation of autophagy. Finally, in Chapter 3, I present our hypothesized model of neutral lipid metabolism process and lipid droplet degradation via mTOR regulating genes in macrophages as well as project conclusion and future directions