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

Richard T. Timms; Sam A. Menzies; Iva A. Tchasovnikarova; Lea Cecilie Christensen; James C. Williamson; Robin Antrobus; Gordon Dougan; Lars Ellgaard; Paul J. Lehner

doi:10.1038/ncomms11786

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

Richard T. Timms, Sam A. Menzies, Iva A. Tchasovnikarova, Lea Cecilie Christensen, James C. Williamson, Robin Antrobus, Gordon Dougan, Lars Ellgaard, Paul J. Lehner

Biomolecular Sciences

37 Citationer (Scopus)

390 Downloads (Pure)

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.

Originalsprog	Engelsk
Artikelnummer	11786
Tidsskrift	Nature Communications
Vol/bind	7
Antal sider	10
ISSN	2041-1723
DOI	https://doi.org/10.1038/ncomms11786
Status	Udgivet - 10 jun. 2016

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10.1038/ncomms11786Licens: CC BY

Timms_2016_Genetic_dissectionForlagets udgivne version, 6,06 MBLicens: CC BY

Citationsformater

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title = "Genetic dissection of mammalian ERAD through comparative haploid and CRISPR forward genetic screens",

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 {\textquoteleft}gold standard{\textquoteright} 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.",

author = "Timms, {Richard T.} and Menzies, {Sam A.} and Tchasovnikarova, {Iva A.} and Christensen, {Lea Cecilie} and Williamson, {James C.} and Robin Antrobus and Gordon Dougan and Lars Ellgaard and Lehner, {Paul J.}",

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T1 - Genetic dissection of mammalian ERAD through comparative haploid and CRISPR forward genetic screens

AU - Timms, Richard T.

AU - Menzies, Sam A.

AU - Tchasovnikarova, Iva A.

AU - Christensen, Lea Cecilie

AU - Williamson, James C.

AU - Antrobus, Robin

AU - Dougan, Gordon

AU - Ellgaard, Lars

AU - Lehner, Paul J.

PY - 2016/6/10

Y1 - 2016/6/10

N2 - 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.

AB - 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.

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DO - 10.1038/ncomms11786

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SN - 2041-1723

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JO - Nature Communications

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