Abstract Over a thousand human genes have dedicated roles in the ubiquitin pathway, making it one of the most complex signaling systems. About 650 of these genes encode ubiquitin ligases (E3s), which recognize substrates and target them for degradation. Our laboratory has developed several methods, based on both cell biological and affinity approaches, to identify substrates of ubiquitin ligases, and have used these to identify over two dozen substrates of five ligases. However, like phosphorylation, in many cases loss of ubiquitination has no obvious phenotype. We have used the R35 mechanism to completely re-configure ourselves into a mammalian cell laboratory and undertaken a large screen to identify phenotypes for poorly understood human E3s/DUBs. We created a CRISPR library against human E3s/DUBs and performed a pooled CRISPR-Cas9 screen combined with chemical inhibition of 41 compounds targeting genome integrity, cell cycle progression, transcription, RNA processing, translation, mitochondrial function, protein folding, metabolic pathways, transport, cytoskeleton, etc. By probing a diverse set of biological processes for E3/DUB involvement, we were able to assess the specificity of these interactions. Overall, we identified one or more specific interactions for 161 E3/DUBs (about 25% of those examined), many of which were previously unstudied. Some genes, such as FBXW7, showed interactions with more than one-third of the compounds. Others showed interactions only with a single compound or a set of related compounds. We are focusing our efforts on following up four sets of ligases: mutants in the poorly studied RING ligase RNF25 were extremely sensitive to alkylating agents, but not other forms of DNA damage; mutants in the unstudied CUL5 adaptor WSB2 were exquisitely sensitive to inhibitors of nuclear export; the CUL4 adaptor DCAF7 has a role in maintaining viability during a CDK4/6 arrest; and the poorly-characterized F-box protein FBXO42 has clear roles in mitosis. We will use both genetic screens and our established mass spec approaches to identify the relevant substrates of these ligases. In addition, we will continue with our analysis of ubiquitin linkages. We have carried out a large genetic interaction screen in yeast to identify roles of individual ubiquitin linkages. We will expand upon this screen and will carry out complementary mass spectrometry approaches to identify relevant substrates.