Cell
Volume 167, Issue 7, 15 December 2016, Pages 1867-1882.e21
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A Multiplexed Single-Cell CRISPR Screening Platform Enables Systematic Dissection of the Unfolded Protein Response

https://doi.org/10.1016/j.cell.2016.11.048Get rights and content
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Highlights

  • Perturb-seq allows parallel screening with rich phenotypic output from single cells

  • Simultaneous delivery and identification of up to three CRISPR perturbations

  • Genome-scale screens dissect the mammalian unfolded protein response

  • Analytical methods separate perturbation responses from confounding effects

Summary

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

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

Cited by (0)

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Co-first authors

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Present address: The Lautenberg Center for General and Tumor Immunology, The BioMedical Research Institute Israel-Canada of the Faculty of Medicine (IMRIC), The Hebrew University Hadassah Medical School, 91120 Jerusalem, Israel

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Present address: New England BioLabs, Ipswich, MA 01938, USA

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Lead contact