Genetic dissection of mammalian ERAD through comparative haploid and CRISPR forward genetic screens
Menzies, Sam A
Christensen, Lea C
Nature Publishing Group
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Timms, R., Menzies, S. A., Tchasovnikarova, I., Christensen, L. C., Williamson, J., Antrobus, R., Dougan, G., et al. (2016). Genetic dissection of mammalian ERAD through comparative haploid and CRISPR forward genetic screens. Nature Communications, 7 (11786)https://doi.org/10.1038/ncomms11786
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 disulfide reductase. Genome-wide CRISPR/Cas9-mediated screens together with haploid genetic screens provide a powerful addition to the forward genetic toolbox.
This work was supported by the Wellcome Trust, through a Principal Research Fellowship to P.J.L. and PhD studentships to S.A.M. and I.A.T., the NIHR Cambridge BRC and the Lundbeck Foundation (L.C.C. and L.E.). The CIMR is in receipt of a Wellcome Trust strategic award.
Wellcome Trust (101835/Z/13/Z)
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External DOI: https://doi.org/10.1038/ncomms11786
This record's URL: https://www.repository.cam.ac.uk/handle/1810/254870