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

TitleA Multiplexed Single-Cell CRISPR Screening Platform Enables Systematic Dissection of the Unfolded Protein Response.
Publication TypeJournal Article
Year of Publication2016
AuthorsAdamson, B, Norman, TM, Jost, M, Cho, MY, Nuñez, JK, Chen, Y, Villalta, JE, Gilbert, LA, Horlbeck, MA, Hein, MY, Pak, RA, Gray, AN, Gross, CA, Dixit, A, Parnas, O, Regev, iv, A, Weissman, JS
JournalCell
Volume167
Issue7
Pagination1867-1882.e21
Date Published2016 Dec 15
ISSN1097-4172
KeywordsAnimals, Clustered Regularly Interspaced Short Palindromic Repeats, Endoribonucleases, Feedback, Humans, Models, Molecular, Protein-Serine-Threonine Kinases, RNA, Guide, Sequence Analysis, RNA, Single-Cell Analysis, Transcription, Genetic, Unfolded Protein Response
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.

DOI10.1016/j.cell.2016.11.048
Alternate JournalCell
PubMed ID27984733
PubMed Central IDPMC5315571
Grant ListU01 CA168370 / CA / NCI NIH HHS / United States
T32 GM007618 / GM / NIGMS NIH HHS / United States
F32 GM116331 / GM / NIGMS NIH HHS / United States
R35 GM118061 / GM / NIGMS NIH HHS / United States
K99 CA204602 / CA / NCI NIH HHS / United States
T32 EB009383 / EB / NIBIB NIH HHS / United States
P50 GM102706 / GM / NIGMS NIH HHS / United States
R01 DA036858 / DA / NIDA NIH HHS / United States
/ / Howard Hughes Medical Institute / United States
R01 AG041826 / AG / NIA NIH HHS / United States
P50 HG006193 / HG / NHGRI NIH HHS / United States
R01 GM102790 / GM / NIGMS NIH HHS / United States
RM1 HG006193 / HG / NHGRI NIH HHS / United States