Document Type
Dissertation - Open Access
Award Date
2023
Degree Name
Doctor of Philosophy (PhD)
Department / School
Biology and Microbiology
First Advisor
Natalie Thiex
Abstract
Advances in CRISPR/Cas9 gene editing technologies present opportunities to better understand the contribution of individual genes to complex eukaryotic cellular processes. I have applied CRISPR whole genome screen technologies to inform further mechanistic understanding of macrophage fitness and neutral lipid metabolism. This work is important because macrophages play critical roles in immune function and are implicated as causative agents in disease states. Chapter One of this dissertation provides insights into how CRISPR genome screening technologies have improved biological discovery and key findings from their application and discusses how this technology can be implemented to advance the discovery of novel macrophage biology. Chapter Two reports the results of CRISPR whole genome screens that identified novel positive and negative regulators of macrophage fitness. There is limited understanding of regulators of macrophage fitness, including mechanisms of survival, proliferation, and adhesion. The screens uncovered p21-activated kinase-2 (PAK2) as a novel suppressor of macrophage fitness. We show that Pak2 gene disruption led to those mutants becoming more proliferative via NF2/Merlin-mediated downregulation of cyclin-D1. In addition, this work indicates that PAK2 suppresses macropinocytic uptake by modulation of actin dynamics. In Chapter Three, I applied CRISPR/Cas9 screening technology to identify critical genes for modified LDL-derived neutral lipid metabolism in macrophages. This is important because macrophages are contributors to atherosclerotic plaque formation. We identified lysosomal nutrient sensing, mTOR signaling, and autophagy as critical pathways and processes regulating neutral lipid content in macrophages. This work provides evidence that lipophagy, the autophagy-mediated degradation of lipid droplets, is the dominant mechanism promoting lipid droplet clearance and supporting cholesterol efflux in macrophages. Chapter Four of this dissertation contextualizes how the findings from the CRISPR whole-genome screens from the preceding chapters contribute to the advancement of macrophage biology and how the findings of this dissertation inform current macrophage-based therapeutical approaches. Chapter Four provides examples of existing approaches where macrophages are therapeutically targeted, their effectiveness, and challenges. Overall, the work presented in this dissertation will serve as a valuable source to inform continued hypothesis generation from unexplored hits and continue to expand our understanding of macrophage biology.
Library of Congress Subject Headings
CRISPR (Genetics)
Macrophages.
Genetic screening.
Publisher
South Dakota State University
Recommended Citation
Wanniarachchi, Kevin, "Application of CRISPR/Cas9 Whole Genome Screens to Advance Macrophage Biology" (2023). Electronic Theses and Dissertations. 848.
https://openprairie.sdstate.edu/etd2/848
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