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

Nat Commun. 2016 Jun 10:7:11786. doi: 10.1038/ncomms11786.

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.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

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

Substances

  • Fluorescent Dyes
  • Glycoproteins
  • Histocompatibility Antigens Class I
  • Thioredoxins
  • alpha-Glucosidases