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Cell. 2016 Dec 15;167(7):1867-1882.e21. doi: 10.1016/j.cell.2016.11.048.

A Multiplexed Single-Cell CRISPR Screening Platform Enables Systematic Dissection of the Unfolded Protein Response.

Author information

  • 1Department of Cellular & Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA; Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, CA 94158, USA; California Institute for Quantitative Biomedical Research, University of California, San Francisco, San Francisco, CA 94158, USA; Center for RNA Systems Biology, University of California, San Francisco, San Francisco, CA 94158, USA.
  • 2Department of Cellular & Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA; Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, CA 94158, USA; California Institute for Quantitative Biomedical Research, University of California, San Francisco, San Francisco, CA 94158, USA; Center for RNA Systems Biology, University of California, San Francisco, San Francisco, CA 94158, USA; Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA 94158, USA.
  • 3Department of Cellular & Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA; Innovative Genomics Initiative, University of California, Berkeley, Berkeley, CA 94720, USA.
  • 4Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA 94158, USA.
  • 5Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA 94158, USA; Department of Cell and Tissue Biology, University of California, San Francisco, San Francisco, CA 94158, USA; Integrative Program in Quantitative Biology, University of California, San Francisco, San Francisco, CA 94158, USA.
  • 6Harvard-MIT Division of Health Sciences and Technology, Cambridge, MA 02142, USA; Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA.
  • 7Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA.
  • 8Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Howard Hughes Medical Institute, Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02140, USA.
  • 9Department of Cellular & Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA; Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, CA 94158, USA; California Institute for Quantitative Biomedical Research, University of California, San Francisco, San Francisco, CA 94158, USA; Center for RNA Systems Biology, University of California, San Francisco, San Francisco, CA 94158, USA. Electronic address: jonathan.weissman@ucsf.edu.

Abstract

Functional genomics efforts face tradeoffs between number of perturbations examined and complexity of phenotypes measured. We bridge this gap with Perturb-seq, which combines droplet-based single-cell RNA-seq with a strategy for barcoding CRISPR-mediated perturbations, allowing many perturbations to be profiled in pooled format. We applied Perturb-seq to dissect the mammalian unfolded protein response (UPR) using single and combinatorial CRISPR perturbations. Two genome-scale CRISPR interference (CRISPRi) screens identified genes whose repression perturbs ER homeostasis. Subjecting ∼100 hits to Perturb-seq enabled high-precision functional clustering of genes. Single-cell analyses decoupled the three UPR branches, revealed bifurcated UPR branch activation among cells subject to the same perturbation, and uncovered differential activation of the branches across hits, including an isolated feedback loop between the translocon and IRE1α. These studies provide insight into how the three sensors of ER homeostasis monitor distinct types of stress and highlight the ability of Perturb-seq to dissect complex cellular responses.

KEYWORDS:

CRIPSRi; CRISPR; Single-cell RNA-seq; cell-to-cell heterogeneity; genome-scale screening; single-cell genomics; unfolded protein response

PMID:
27984733
PMCID:
PMC5315571
[Available on 2017-12-15]
DOI:
10.1016/j.cell.2016.11.048
[PubMed - in process]
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