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


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Authors

Menzies, Sam A 
Tchasovnikarova, Iva A  ORCID logo  https://orcid.org/0000-0002-0477-0956
Christensen, Lea C 
Williamson, James C 

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.

Description

Keywords

Animals, CRISPR-Cas Systems, Endoplasmic Reticulum-Associated Degradation, Fluorescent Dyes, Genes, Reporter, Genetic Testing, Glycoproteins, HEK293 Cells, Haploidy, HeLa Cells, Histocompatibility Antigens Class I, Humans, Mammals, Oxidation-Reduction, Protein Binding, Protein Domains, Thioredoxins, alpha-Glucosidases

Journal Title

Nat Commun

Conference Name

Journal ISSN

2041-1723
2041-1723

Volume Title

7

Publisher

Springer Science and Business Media LLC
Sponsorship
Wellcome Trust (101835/Z/13/Z)
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.