Year: 2020
Type: dissertation
Abstract: Over 7,000 rare diseases have been identified in the human population that, in aggregate, affect over 400 million people worldwide. The majority of rare diseases that have been identified are genetic in origin. However, a key challenge in developing therapies is that the functions of many rare disease-associated genes are poorly understood and consequently, their role in disease pathogenesis. There is strong appreciation that genes do not act in isolation and many disease phenotypes may arise from complex genetic interactions (GIs). Systematic mapping of GIs offers a wealth of information about gene function and advancements in gene-editing technology, such as clustered regularly interspaced short palindromic repeats (CRISPR), allows for GI mapping in human cells. In this thesis, I describe how lentiviral-based genome-wide pooled CRISPR/Cas9 screens offer insight into the function of rare disease-associated genes including NGLY1 and the genes encoding the complement regulatory proteins (CRPs): CD46, CD55, and CD59. Autosomal recessive mutations in NGLY1 give rise to NGLY1-deficiency, a congenital disorder of deglycosylation. How the loss of NGLY1 function contributes to disease pathogenesis is poorly understood. The CRPs are key inhibitors of the complement cascade and their impaired function contributes to a wide range of common and rare diseases. Similar to NGLY1, the functions of the CRPs independent of complement-regulation and roles in disease have not been explored using functional genomics approaches. To further our functional understanding of NGLY1 and the genes encoding the CRPs, I performed pooled genome-wide CRISPR/Cas9 screens to identify GIs, and uncovered a novel genetic relationship between the CRPs and the NGLY1 pathway involving genes in the secretory pathway that may coordinate different aspects of intracellular cholesterol trafficking. My work demonstrates the utility in mapping GIs in order to reveal functional relationships with rare disease genes, offering critical insight into the genetic underpinnings of NGLY1-deficiency and complement-independent functions of the CRPs. As the functions of many rare disease-associated genes are poorly characterized, mapping genetic interactions in co-isogenic human cell lines using genome-wide CRISPR screening approaches offers a powerful framework that may be exploited to gain insight into gene function and direct future therapeutic efforts.
Source: TSpace (University of Toronto)
Author: Kristin Kantautas
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W3105893881