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Using CRISPR/Cas9 to Identify Gene Interactions with Hexosamine Biosynthesis and N-Glycan Remodeling Pathway Enzymes

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Using CRISPR/Cas9 to Identify Gene Interactions with Hexosamine Biosynthesis and N-Glycan Remodeling Pathway Enzymes Using CRISPR/Cas9 to Identify Gene Interactions with Hexosamine Biosynthesis and N-Glycan Remodeling Pathway Enzymes by Alexandra Chirila A thesis submitted in conformity with the requirements for the degree of Master of Science Laboratory Medicine and Pathobiology University of Toronto © Copyright by Alexandra Chirila 2019 Using CRISPR/Cas9 to Identify Gene Interactions with Hexosamine Biosynthesis and N-Glycan Remodeling Pathway Enzymes Alexandra Chirila Master of Science Laboratory Medicine and Pathobiology University of Toronto 2019 Abstract Genetic studies by classical mutagenesis and screening methods have revealed many molecular interactions and regulatory relationships in animal models. The hexosamine biosynthesis pathway and N-glycosylation are upregulated in most cancers and have shown to play a role in many cancer cell phenotypes. In this thesis, a CRISPR/Cas9 genome-wide targeted mutagenesis approach was employed to identify gene interactions with chosen genes-of-interest from these two pathways: NAGK, GFPT1, MGAT1 and MGAT5. The gene interactions identified suggest relationships between our genes-of-interest and cell-cell adhesion, cytoskeleton, and folate and nucleotide metabolism. Further characterization of metabolite levels in the gene-of-interest knockout cells was done to help understand potential gene interactions from the screen. For example, metabolic imbalance in mutant cells likely indicates cell stress and reactive oxygen species, consistent with PRDX1, an antioxidant, being a suggested genetic interactor in multiple screens. These findings provide new insight on vulnerabilities and genomic redundancies in cancer cells. ii Acknowledgments I would first like to thank my supervisor, Dr. James Dennis, for the continuous mentorship and support throughout my master’s thesis project. He was always willing to provide guidance and share his immense knowledge, and was very patient and enthusiastic. I would also like to thank Dr. Payman Tehrani, Dr. Michael Aregger, and Dr. Keith Lawson for much guidance and assistance throughout my thesis project. I thank all members of the Dennis/Swallow lab for the discussions, support and assistance. I would also like to thank my advisory committee: Dr. Linda Penn and Dr. Jason Moffat, for providing me with direction and expanding my knowledge on my project, and Dr. Irene Andrulis for sitting on my examination committee. iii Contributions The author performed all experiments described in this thesis with the following contributions: Jason Moffat’s Lab: Conducted multiple HAP1 WT genome-wide CRISPR/Cas9 KO screens for comparison to the KO screen data (prior to my project start date). Michael Aregger in Jason Moffat’s Lab: Conducted the HAP1 NAGK KO genome-wide CRISPR/Cas9 KO screen (prior to my project start date). Aldis Krizus in Jim Dennis’s Lab: Generated the MDA-MB-231 NAGK, GNPNAT1, MGAT1 and MGAT5 KO cell lines, and also conducted the sample preparation for the MDA-MB-231 metabolomics experiment outlined in Figure 4.1. Katie Chan in Jason Moffat’s Lab: Made the virus containing the Toronto KnockOut CRISPR Library - Version 3 (TKOv3) for all of the CRISPR/Cas9 KO screens. Amy Tong in Jason Moffat’s Lab: Coordinated sequencing library preparations and managed sequence data for all of the CRISPR/Cas9 KO screens. Max Billmann in Jason Moffat’s Lab: Conducted sequence analysis for all of the CRISPR/Cas9 KO screens up to generation of pi-scores and False Discovery Rates (FDRs). Michael Parsons in the Lunenfeld-Tanenbaum Research Institute: Assisted with flow cytometry analysis. Judy Pawling in Jim Dennis’s Lab: Conducted the metabolomics sample preparation beyond the flash freezing in liquid nitrogen, ran the samples through liquid chromatography-tandem mass spectrometry, and analyzed the raw data generating expression values normalized to cell number for each cell line. Judy also conducted the MDA-MB-231 in vivo tumor xenograft experiment in NOD-SCID mice outlined in Figure 1.1 with Karina Pacholczyk, and also generated Figures 3.12 and 3.13. Karina Pacholczyk in Dennis’s Lab: Conducted the MDA-MB-231 in vivo tumor xenograft experiment in NOD-SCID mice outlined in Figure 1.1 with Judy Pawling. iv Table of Contents Abstract .................................................................................................................................. ii Acknowledgments ................................................................................................................. iii Contributions ......................................................................................................................... iv List of Tables ......................................................................................................................... vii List of Figures ....................................................................................................................... viii List of Appendices ................................................................................................................... x List of Abbreviations .............................................................................................................. xi Chapter 1 ................................................................................................................................ 1 Introduction .................................................................................................................... 1 1.1 Protein N-Glycosylation as a Known-Vulnerability ................................................................ 2 1.2 Hexosamine Biosynthesis Pathway ...................................................................................... 7 1.2.1 Glycosylation ......................................................................................................................................... 9 1.3 Cancer Cell Metabolism ...................................................................................................... 13 1.3.1 Main Cancer Energy Sources ............................................................................................................... 15 1.3.2 Increased flux through HBP ................................................................................................................ 17 1.4 Gene Editing ...................................................................................................................... 18 1.4.1 CRISPR/Cas9 ........................................................................................................................................ 20 1.5 Genetic Interactions ........................................................................................................... 21 1.5.1 Global gene interaction network ........................................................................................................ 23 1.5.2 Yeast versus Human Genome ............................................................................................................. 24 1.6 Rationale ........................................................................................................................... 25 Chapter 2 .............................................................................................................................. 28 Materials and Methods ................................................................................................. 28 2.1 Materials ........................................................................................................................... 28 2.2 MethoDs ............................................................................................................................ 33 v Chapter 3 .............................................................................................................................. 46 Results .......................................................................................................................... 46 3.1 HAP1 CRISPR/Cas9 Screens ................................................................................................ 46 3.2 Validation Competition Assay ............................................................................................ 62 3.3 Metabolomics .................................................................................................................... 68 Chapter 4 .............................................................................................................................. 76 Discussion ..................................................................................................................... 76 4.1 HAP1 CRISPR/Cas9 Screens ................................................................................................ 76 4.2 Validation Competition Assay ............................................................................................ 78 4.3 Metabolomics .................................................................................................................... 80 4.4 Limitations ......................................................................................................................... 86 4.5 Future Directions ............................................................................................................... 88 4.6 Conclusions ........................................................................................................................ 89 References ...........................................................................................................................
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