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Targeted protein degradation technologies employ small-molecule ‘degraders’ — proteolysis-targeting chimeras (PROTACs) or monovalent molecular glues – that form a ternary complex with a protein of interest and an E3 ubiquitin ligase (typically von Hippel-Lindau (VHL) or cereblon (CRBN)), resulting in target ubiquitination and subsequent proteasomal degradation. Depending on the degrader and target protein, different E3 interfaces — or ‘functional hotspots’ — are critically involved. Identifying such hotspots could inform on strategies to optimize degrader design and mechanisms of degrader resistance. Here, using human haploid genetics and deep mutational scanning (DMS), Hanzl et al. locate functional VHL and CRBN hotspots that are of general relevance, or specific for different degraders and target proteins. Hotspot mutations were found to converge on altered ternary-complex assemblies. Integration of DMS data with available clinical data highlighted a number of the identified CRBN hotspots to be disrupted by mutations in multiple myeloma patients relapsing from treatment with CRBN-based molecular glues.