Colorectal cancer (CRC) is the second leading cause of cancer-related mortality in Canada. These statistics underscore the need to develop more effective therapies, which requires a deeper understanding of CRC pathogenesis. Chromosome instability (CIN) is characterized by an increased rate of chromosome gains and losses and occurs in approximately 85% of CRC cases. However, the aberrant genes driving CIN (i.e., CIN genes) are largely unknown. Emerging data suggest ubiquitin homeostasis is crucial for maintaining chromosome stability, and ubiquitylation and deubiquitylation genes undergo copy number losses in CRC. Thus, I hypothesized that diminished expression of ubiquitylation and deubiquitylation genes induce CIN and cellular transformation, promoting CRC development. This thesis utilized an established research pipeline coupling gene silencing with quantitative imaging microscopy (QuantIM) to investigate 581 ubiquitylation and 94 deubiquitylation genes for impacts on CIN across three karyotypically stable cell lines (HCT116, 1CT and hTERT) (Chapter 4). This screen assessed CIN-associated phenotypes, including changes in nuclear areas and increases in micronucleus formation. Subsequent direct tests in non-malignant colonic epithelial cell lines confirmed that silencing 10 prioritized genes, including USP4 and SKP2, induces significant changes in chromosome numbers and thus, CIN. As USP4 copy number losses occur in ~16% of CRC cases and correspond with worse patient outcomes relative to diploid counterparts, heterozygous and homozygous CRISPR/Cas9 USP4-knockout clones were generated in two non-malignant colonic epithelial cell lines (Chapter 5). Although these knockout clones did not form subcutaneous tumours in mice within the constraints of this study, they exhibited dynamic CIN and cellular transformation phenotypes, suggesting USP4 copy number loss may be an early etiological event in CRC pathogenesis. Parallel research in our laboratory showed that reduced SKP2 expression induces CIN through mechanisms not fully explained by the established substrates Cyclin E1 and P27. To expand these findings, TurboID was employed, which identified the actin-binding proteins Caldesmon and Transgelin as putative SKP2 substrates (Chapter 6). These data implicate novel roles for SKP2 in modulating actin dynamics that may impact chromosome stability. Collectively, these findings provide novel insights into how aberrant ubiquitin regulation impacts CIN and may contribute to CRC pathogenesis.