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A comprehensive phenotypic CRISPR-Cas9 screen of the ubiquitin pathway uncovers roles of ubiquitin ligases in mitosis


The complex human ubiquitin proteasome system, comprised of over 700 ubiquitin ligases (E3s) and deubiquitinases (DUBs), has been difficult to systematically and phenotypically characterize. We performed a chemical-genetic CRISPR-Cas9 screen to identify E3s/DUBs whose loss renders cells sensitive or resistant to 41 compounds targeting a broad range of biology, including cytoskeletal integrity, mitosis, G1/S progression, genome stability, translation, metabolism, and vesicular transport.  Genes and compounds both clustered functionally, showing that inhibitors of related pathways interact similarly with the E3s/DUBs and demonstrating the robustness of our screen. Some genes, such as FBXW7, showed interactions with more than one-third of the compounds. Others, such as the mostly un-studied RNF25 and FBXO42, showed interactions primarily with a single compound (MMS for RNF25) or a set of related compounds (mitotic cluster for FBXO42). Mutation of several E3s with sensitivity to mitotic inhibitors had an increase in aberrant mitoses, suggesting a role for these genes in cell cycle regulation. Overall, our comprehensive CRISPR-Cas9 screen uncovered 466 gene-compound interactions covering 25% of the E3s/DUBs interrogated.

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