Targeted protein degradation (TPD) is a rapidly developing field that hijacks the cell’s ubiquitin-proteasome system by using small molecules to induce proximity between a protein of interest and an E3 ubiquitin ligase, leading to target ubiquitination and subsequent proteasomal degradation. These TPD-inducing molecules, termed degraders, offer advantages over traditional occupancy-based inhibitors by operating through event-driven pharmacology, making this modality attractive as chemical biology tools and therapeutics. Molecular glues represent a unique class of degraders due to their monovalent nature and favorable drug-like properties, making them attractive both as therapeutics and as scaffolds for the development of heterobifunctional degraders. These molecules are also uniquely selective due to the high degree of protein and ligand cooperativity required for productive ternary complex formation between the neosubstrate and the E3 ubiquitin ligase. However, the rational development of glue degraders has been challenging, with most known examples identified through serendipitous discovery or highthroughput screening. Thus, strategies that enable the elucidation of their structural prerequisites, E3 ligase scaffold preferences, and potential resistance mechanisms will be important for accelerating the discovery and design of molecular glue degraders. In this thesis, I present three projects that leverage functional genomics approaches, including CRISPR-suppressor screens, base editor scanning, and deep mutational scanning, to illuminate molecular principles governing TPD by molecular glue degraders. Genome editing technologies enable systematic amino acid-resolution mutagenesis in endogenous contexts, iv while expression library screens allow comprehensive evaluation of genetic variation in a controlled, unbiased manner to map structure-function relationships of E3 ligases. Together, these approaches reveal novel insights into mechanisms of action, resistance, and chemicalgenetic convergence, which can be harnessed to guide rational degrader design. In Chapter 2, I describe our efforts to map the landscape of resistance mutations in the neosubstrates RBM39 and GSPT1 in response to their respective molecular glue degraders. Through CRISPR-Cas9 tiling screens, we uncover canonical degron mutations that disrupt degrader binding, as well as distal site mutations that partially rescue degradation yet are sufficient for cell survival. Integration with evolutionary sequence conservation data reveals varying levels of sequence conservation across resistance sites in RBM39 and GSPT1, suggesting that structural and functional requirements of protein regions constrain the accessible mutational space and thereby drive divergent mutational outcomes across targets. In Chapter 3, I focus on elucidating the mechanism of action of the molecular glue degrader UM171, which induces the degradation of the LSD1-CoREST corepressor complex via the Cullin3-RING E3 ligase substrate receptor, KBTBD4. By integrating proteomics, functional genomics, biochemical analysis, and cryo-electron microscopy (cryo-EM), we show that HDAC1/2, the direct target of UM171, bridges CoREST to KBTBD4, and that CoREST degradation requires a dual-glue mechanism involving the metabolite inositol hexakisphosphate (InsP6). Base editor scanning further validates critical residues in the ternary complex interface, showcasing the value of precise genetic perturbations in probing requirements for degrader activity. In Chapter 4, I investigate KBTBD4 mutations found in medulloblastoma, demonstrating that these mutants are gain-of-function (GOF) and mimic UM171 by promoting neomorphic degradation of CoREST through HDAC1/2 engagement. Using deep mutational scanning, we chart the mutational landscape of the KBTBD4 cancer hotspot, revealing a strong preference for insertion mutations in driving neomorphic E3-substrate engagement. Unexpectedly, structural v analyses demonstrate a convergent shape complementarity phenomenon between these cancer mutations and UM171, where genetic mutations and chemical matter structurally and functionally mimic each other. We also demonstrate that HDAC1/2 inhibitors can disrupt the mutant ternary complex and suppress proliferation in patient-derived KBTBD4-mutant medulloblastoma cells, revealing a therapeutically actionable vulnerability. Collectively, these studies illustrate the power of massively parallel genetic screening in uncovering mechanistic principles of molecular glue degraders, identifying resistance vulnerabilities, and guiding degrader design, prompting their use as valuable tools in the TPD space. Importantly, we describe a phenomenon of chemical-genetic convergence where genetic mutations and small molecules phenocopy each other structurally and functionally, highlighting a framework where genetic perturbations can be used to aid the rational design of induced proximity therapeutics. More broadly, this work underscores the potential of functional genomics to accelerate the discovery of next-generation degraders and proximity-based therapeutics.