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

Britt Adamson; Thomas M Norman; Marco Jost; Min Y Cho; James K Nuñez; Yuwen Chen; Jacqueline E Villalta; Luke A Gilbert; Max A Horlbeck; Marco Y Hein; Ryan A Pak; Andrew N Gray; Carol A Gross; Atray Dixit; Oren Parnas; Aviv Regev; Jonathan S Weissman

doi:10.1016/j.cell.2016.11.048

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

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

Authors

Britt Adamson¹, Thomas M Norman¹, Marco Jost², Min Y Cho¹, James K Nuñez¹, Yuwen Chen¹, Jacqueline E Villalta¹, Luke A Gilbert¹, Max A Horlbeck¹, Marco Y Hein¹, Ryan A Pak³, Andrew N Gray⁴, Carol A Gross⁵, Atray Dixit⁶, Oren Parnas⁷, Aviv Regev⁸, Jonathan S Weissman⁹

Affiliations

¹ Department 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.
² Department 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.
³ Department 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.
⁴ Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA 94158, USA.
⁵ Department 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.
⁶ Harvard-MIT Division of Health Sciences and Technology, Cambridge, MA 02142, USA; Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA.
⁷ Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA.
⁸ Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Howard Hughes Medical Institute, Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02140, USA.
⁹ Department 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.

MeSH terms

Animals
Clustered Regularly Interspaced Short Palindromic Repeats
Endoribonucleases
Feedback
Humans
Models, Molecular
Protein Serine-Threonine Kinases
RNA, Guide, CRISPR-Cas Systems / metabolism
Sequence Analysis, RNA / methods*
Single-Cell Analysis / methods*
Transcription, Genetic
Unfolded Protein Response

Substances

RNA, Guide, CRISPR-Cas Systems
ERN1 protein, human
Protein Serine-Threonine Kinases
Endoribonucleases

Abstract

MeSH terms

Substances

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