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 Citations (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.

Original language	English
Article number	11786
Journal	Nature Communications
Volume	7
Number of pages	10
ISSN	2041-1723
DOIs	https://doi.org/10.1038/ncomms11786
Publication status	Published - 10 Jun 2016

Access to Document

10.1038/ncomms11786Licence: CC BY

Timms_2016_Genetic_dissectionFinal published version, 6.06 MBLicence: CC BY

Cite this

@article{8880bd814d8348fc8b76d3ec07986d0a,

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.}",

year = "2016",

month = jun,

day = "10",

doi = "10.1038/ncomms11786",

language = "English",

volume = "7",

journal = "Nature Communications",

issn = "2041-1723",

publisher = "nature publishing group",

}

TY - JOUR

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.

U2 - 10.1038/ncomms11786

DO - 10.1038/ncomms11786

M3 - Journal article

C2 - 27283361

SN - 2041-1723

VL - 7

JO - Nature Communications

JF - Nature Communications

M1 - 11786

ER -