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Genetic dissection of mammalian ERAD through comparative haploid and CRISPR forward genetic screens

Author

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  • Richard T. Timms

    (Cambridge Institute for Medical Research, Cambridge Biomedical Campus)

  • Sam A. Menzies

    (Cambridge Institute for Medical Research, Cambridge Biomedical Campus)

  • Iva A. Tchasovnikarova

    (Cambridge Institute for Medical Research, Cambridge Biomedical Campus)

  • Lea C. Christensen

    (University of Copenhagen)

  • James C. Williamson

    (Cambridge Institute for Medical Research, Cambridge Biomedical Campus)

  • Robin Antrobus

    (Cambridge Institute for Medical Research, Cambridge Biomedical Campus)

  • Gordon Dougan

    (Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus)

  • Lars Ellgaard

    (University of Copenhagen)

  • Paul J. Lehner

    (Cambridge Institute for Medical Research, Cambridge Biomedical Campus)

Abstract

The application of forward genetic screens to cultured human cells represents a powerful method to study gene function. The repurposing of the bacterial CRISPR/Cas9 system provides an effective method to disrupt gene function in mammalian cells, and has been applied to genome-wide screens. Here, we compare the efficacy of genome-wide CRISPR/Cas9-mediated forward genetic screens versus gene-trap mutagenesis screens in haploid human cells, which represent the existing ‘gold standard’ method. This head-to-head comparison aimed to identify genes required for the endoplasmic reticulum-associated degradation (ERAD) of MHC class I molecules. The two approaches show high concordance (>70%), successfully identifying the majority of the known components of the canonical glycoprotein ERAD pathway. Both screens also identify a role for the uncharacterized gene TXNDC11, which we show encodes an EDEM2/3-associated disulphide reductase. Genome-wide CRISPR/Cas9-mediated screens together with haploid genetic screens provide a powerful addition to the forward genetic toolbox.

Suggested Citation

  • Richard T. Timms & Sam A. Menzies & Iva A. Tchasovnikarova & Lea C. Christensen & James C. Williamson & Robin Antrobus & Gordon Dougan & Lars Ellgaard & Paul J. Lehner, 2016. "Genetic dissection of mammalian ERAD through comparative haploid and CRISPR forward genetic screens," Nature Communications, Nature, vol. 7(1), pages 1-10, September.
    • Handle: RePEc:nat:natcom:v:7:y:2016:i:1:d:10.1038_ncomms11786
    DOI: 10.1038/ncomms11786
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    1. Morgane Boone & Pathmanaban Ramasamy & Jasper Zuallaert & Robbin Bouwmeester & Berre Moer & Davy Maddelein & Demet Turan & Niels Hulstaert & Hannah Eeckhaut & Elien Vandermarliere & Lennart Martens & , 2021. "Massively parallel interrogation of protein fragment secretability using SECRiFY reveals features influencing secretory system transit," Nature Communications, Nature, vol. 12(1), pages 1-16, December.
    2. Ian D. Ferguson & Bonell Patiño-Escobar & Sami T. Tuomivaara & Yu-Hsiu T. Lin & Matthew A. Nix & Kevin K. Leung & Corynn Kasap & Emilio Ramos & Wilson Nieves Vasquez & Alexis Talbot & Martina Hale & A, 2022. "The surfaceome of multiple myeloma cells suggests potential immunotherapeutic strategies and protein markers of drug resistance," Nature Communications, Nature, vol. 13(1), pages 1-17, December.
    3. Anna S. Dickson & Tekle Pauzaite & Esther Arnaiz & Brian M. Ortmann & James A. West & Norbert Volkmar & Anthony W. Martinelli & Zhaoqi Li & Niek Wit & Dennis Vitkup & Arthur Kaser & Paul J. Lehner & J, 2023. "A HIF independent oxygen-sensitive pathway for controlling cholesterol synthesis," Nature Communications, Nature, vol. 14(1), pages 1-16, December.

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