Genetic dissection of mammalian ERAD through comparative haploid and CRISPR forward genetic screens.
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.
Department of Medicine, Cambridge Institute for Medical Research, Cambridge Biomedical Campus, Hills Road, Cambridge CB2 0XY, UK.
Richard T
0000-0001-7275-597X
RT
Timms
Department of Medicine, Cambridge Institute for Medical Research, Cambridge Biomedical Campus, Hills Road, Cambridge CB2 0XY, UK.
Sam A
SA
Menzies
Department of Medicine, Cambridge Institute for Medical Research, Cambridge Biomedical Campus, Hills Road, Cambridge CB2 0XY, UK.
Iva A
0000-0002-0477-0956
IA
Tchasovnikarova
Department of Biology, University of Copenhagen, 2200 Copenhagen N, Denmark.
Lea C
LC
Christensen
Department of Medicine, Cambridge Institute for Medical Research, Cambridge Biomedical Campus, Hills Road, Cambridge CB2 0XY, UK.
James C
JC
Williamson
Department of Medicine, Cambridge Institute for Medical Research, Cambridge Biomedical Campus, Hills Road, Cambridge CB2 0XY, UK.
Robin
R
Antrobus
Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Cambridge CB10 1SA, UK.
Gordon
G
Dougan
Department of Biology, University of Copenhagen, 2200 Copenhagen N, Denmark.
Lars
0000-0002-7018-0137
L
Ellgaard
Department of Medicine, Cambridge Institute for Medical Research, Cambridge Biomedical Campus, Hills Road, Cambridge CB2 0XY, UK.
Paul J
PJ
Lehner
D000818:Animals; D064113:CRISPR-Cas Systems / Q000235:genetics*; D060746:Endoplasmic Reticulum-Associated Degradation / Q000235:genetics*; D005456:Fluorescent Dyes / Q000378:metabolism; D017930:Genes, Reporter; D005820:Genetic Testing; D006023:Glycoproteins / Q000378:metabolism; D057809:HEK293 Cells; D006238:Haploidy; D006367:HeLa Cells; D015395:Histocompatibility Antigens Class I / Q000378:metabolism; D006801:Humans; D008322:Mammals / Q000235:genetics*; D010084:Oxidation-Reduction; D011485:Protein Binding; D000072417:Protein Domains; D013879:Thioredoxins / Q000737:chemistry / Q000378:metabolism; D000520:alpha-Glucosidases / Q000378:metabolism
D005456:Fluorescent Dyes; D006023:Glycoproteins; D015395:Histocompatibility Antigens Class I; D013879:Thioredoxins; D000520:alpha-Glucosidases