Perturb-Seq: Dissecting Molecular Circuits with Scalable Single-Cell RNA Profiling of Pooled Genetic Screens

Atray Dixit; Oren Parnas; Biyu Li; Jenny Chen; Charles P. Fulco; Livnat Jerby-Arnon; Nemanja D. Marjanovic; Danielle Dionne; Tyler Burks; Raktima Raychowdhury; Britt Adamson; Thomas M. Norman; Eric S. Lander; Jonathan S. Weissman; Nir Friedman; Aviv Regev

doi:10.1016/j.cell.2016.11.038

Perturb-Seq: Dissecting Molecular Circuits with Scalable Single-Cell RNA Profiling of Pooled Genetic Screens

Atray Dixit, Oren Parnas, Biyu Li, Jenny Chen, Charles P. Fulco, Livnat Jerby-Arnon, Nemanja D. Marjanovic, Danielle Dionne, Tyler Burks, Raktima Raychowdhury, Britt Adamson, Thomas M. Norman, Eric S. Lander, Jonathan S. Weissman, Nir Friedman, Aviv Regev^*

^*Corresponding author for this work

Research output: Contribution to journal › Article › peer-review

803 Scopus citations

Abstract

Genetic screens help infer gene function in mammalian cells, but it has remained difficult to assay complex phenotypes—such as transcriptional profiles—at scale. Here, we develop Perturb-seq, combining single-cell RNA sequencing (RNA-seq) and clustered regularly interspaced short palindromic repeats (CRISPR)-based perturbations to perform many such assays in a pool. We demonstrate Perturb-seq by analyzing 200,000 cells in immune cells and cell lines, focusing on transcription factors regulating the response of dendritic cells to lipopolysaccharide (LPS). Perturb-seq accurately identifies individual gene targets, gene signatures, and cell states affected by individual perturbations and their genetic interactions. We posit new functions for regulators of differentiation, the anti-viral response, and mitochondrial function during immune activation. By decomposing many high content measurements into the effects of perturbations, their interactions, and diverse cell metadata, Perturb-seq dramatically increases the scope of pooled genomic assays.

Original language	American English
Pages (from-to)	1853-1866.e17
Journal	Cell
Volume	167
Issue number	7
DOIs	https://doi.org/10.1016/j.cell.2016.11.038
State	Published - 15 Dec 2016

Bibliographical note

Funding Information:
We thank A. Basu, C. Muus, C. Rodman, and N. Rogel for help in unpublished pilot; D. Feldman for feedback; K. Shekhar for vector design; B. Cleary and A. Bloemendal for discussions on combinatorial models; T. Wang for an unpublished plasmid; L. Gaffney for help with figures; and the Genomics Platform for sequencing. Support was from NDSEG Fellowship (A.D.), NIH T32GM87232 (N.M.), Klarman Cell Observatory (A.R.), NHGRI RM1HG006193 (N.F., A.R.), and HHMI (A.R.). J.S.W. is a founder of KSQ Therapeutics, a CRISPR functional genomics company. J.S.W. has filed a patent application related to CRISPRi and CRISPRa screening (PCT/US15/40449), and A.D., O.P., B.A., T.M.N., E.S.L., J.S.W., and A.R. have filed a patent application related to Perturb-seq. A.R. is an SAB member of ThermoFisher, Syros, and Driver. The Broad Institute, which E.S.L. directs, holds patents and has filed patent applications on technologies related to other aspects of CRISPR.

Publisher Copyright:
© 2016 Elsevier Inc.

Keywords

CRISPR
epistasis
genetic interactions
pooled screen
single-cell RNA-seq

Access to Document

10.1016/j.cell.2016.11.038

Cite this

Dixit, A., Parnas, O., Li, B., Chen, J., Fulco, C. P., Jerby-Arnon, L., Marjanovic, N. D., Dionne, D., Burks, T., Raychowdhury, R., Adamson, B., Norman, T. M., Lander, E. S., Weissman, J. S., Friedman, N., & Regev, A. (2016). Perturb-Seq: Dissecting Molecular Circuits with Scalable Single-Cell RNA Profiling of Pooled Genetic Screens. Cell, 167(7), 1853-1866.e17. https://doi.org/10.1016/j.cell.2016.11.038

@article{57c4633b60c24f48abb49bd805afcd07,

title = "Perturb-Seq: Dissecting Molecular Circuits with Scalable Single-Cell RNA Profiling of Pooled Genetic Screens",

abstract = "Genetic screens help infer gene function in mammalian cells, but it has remained difficult to assay complex phenotypes—such as transcriptional profiles—at scale. Here, we develop Perturb-seq, combining single-cell RNA sequencing (RNA-seq) and clustered regularly interspaced short palindromic repeats (CRISPR)-based perturbations to perform many such assays in a pool. We demonstrate Perturb-seq by analyzing 200,000 cells in immune cells and cell lines, focusing on transcription factors regulating the response of dendritic cells to lipopolysaccharide (LPS). Perturb-seq accurately identifies individual gene targets, gene signatures, and cell states affected by individual perturbations and their genetic interactions. We posit new functions for regulators of differentiation, the anti-viral response, and mitochondrial function during immune activation. By decomposing many high content measurements into the effects of perturbations, their interactions, and diverse cell metadata, Perturb-seq dramatically increases the scope of pooled genomic assays.",

keywords = "CRISPR, epistasis, genetic interactions, pooled screen, single-cell RNA-seq",

author = "Atray Dixit and Oren Parnas and Biyu Li and Jenny Chen and Fulco, {Charles P.} and Livnat Jerby-Arnon and Marjanovic, {Nemanja D.} and Danielle Dionne and Tyler Burks and Raktima Raychowdhury and Britt Adamson and Norman, {Thomas M.} and Lander, {Eric S.} and Weissman, {Jonathan S.} and Nir Friedman and Aviv Regev",

note = "Funding Information: We thank A. Basu, C. Muus, C. Rodman, and N. Rogel for help in unpublished pilot; D. Feldman for feedback; K. Shekhar for vector design; B. Cleary and A. Bloemendal for discussions on combinatorial models; T. Wang for an unpublished plasmid; L. Gaffney for help with figures; and the Genomics Platform for sequencing. Support was from NDSEG Fellowship (A.D.), NIH T32GM87232 (N.M.), Klarman Cell Observatory (A.R.), NHGRI RM1HG006193 (N.F., A.R.), and HHMI (A.R.). J.S.W. is a founder of KSQ Therapeutics, a CRISPR functional genomics company. J.S.W. has filed a patent application related to CRISPRi and CRISPRa screening (PCT/US15/40449), and A.D., O.P., B.A., T.M.N., E.S.L., J.S.W., and A.R. have filed a patent application related to Perturb-seq. A.R. is an SAB member of ThermoFisher, Syros, and Driver. The Broad Institute, which E.S.L. directs, holds patents and has filed patent applications on technologies related to other aspects of CRISPR. Publisher Copyright: {\textcopyright} 2016 Elsevier Inc.",

year = "2016",

month = dec,

day = "15",

doi = "10.1016/j.cell.2016.11.038",

language = "American English",

volume = "167",

pages = "1853--1866.e17",

journal = "Cell",

issn = "0092-8674",

publisher = "Cell Press",

number = "7",

}

Dixit, A, Parnas, O, Li, B, Chen, J, Fulco, CP, Jerby-Arnon, L, Marjanovic, ND, Dionne, D, Burks, T, Raychowdhury, R, Adamson, B, Norman, TM, Lander, ES, Weissman, JS, Friedman, N & Regev, A 2016, 'Perturb-Seq: Dissecting Molecular Circuits with Scalable Single-Cell RNA Profiling of Pooled Genetic Screens', Cell, vol. 167, no. 7, pp. 1853-1866.e17. https://doi.org/10.1016/j.cell.2016.11.038

TY - JOUR

T1 - Perturb-Seq

T2 - Dissecting Molecular Circuits with Scalable Single-Cell RNA Profiling of Pooled Genetic Screens

AU - Dixit, Atray

AU - Parnas, Oren

AU - Li, Biyu

AU - Chen, Jenny

AU - Fulco, Charles P.

AU - Jerby-Arnon, Livnat

AU - Marjanovic, Nemanja D.

AU - Dionne, Danielle

AU - Burks, Tyler

AU - Raychowdhury, Raktima

AU - Adamson, Britt

AU - Norman, Thomas M.

AU - Lander, Eric S.

AU - Weissman, Jonathan S.

AU - Friedman, Nir

AU - Regev, Aviv

N1 - Funding Information: We thank A. Basu, C. Muus, C. Rodman, and N. Rogel for help in unpublished pilot; D. Feldman for feedback; K. Shekhar for vector design; B. Cleary and A. Bloemendal for discussions on combinatorial models; T. Wang for an unpublished plasmid; L. Gaffney for help with figures; and the Genomics Platform for sequencing. Support was from NDSEG Fellowship (A.D.), NIH T32GM87232 (N.M.), Klarman Cell Observatory (A.R.), NHGRI RM1HG006193 (N.F., A.R.), and HHMI (A.R.). J.S.W. is a founder of KSQ Therapeutics, a CRISPR functional genomics company. J.S.W. has filed a patent application related to CRISPRi and CRISPRa screening (PCT/US15/40449), and A.D., O.P., B.A., T.M.N., E.S.L., J.S.W., and A.R. have filed a patent application related to Perturb-seq. A.R. is an SAB member of ThermoFisher, Syros, and Driver. The Broad Institute, which E.S.L. directs, holds patents and has filed patent applications on technologies related to other aspects of CRISPR. Publisher Copyright: © 2016 Elsevier Inc.

PY - 2016/12/15

Y1 - 2016/12/15

N2 - Genetic screens help infer gene function in mammalian cells, but it has remained difficult to assay complex phenotypes—such as transcriptional profiles—at scale. Here, we develop Perturb-seq, combining single-cell RNA sequencing (RNA-seq) and clustered regularly interspaced short palindromic repeats (CRISPR)-based perturbations to perform many such assays in a pool. We demonstrate Perturb-seq by analyzing 200,000 cells in immune cells and cell lines, focusing on transcription factors regulating the response of dendritic cells to lipopolysaccharide (LPS). Perturb-seq accurately identifies individual gene targets, gene signatures, and cell states affected by individual perturbations and their genetic interactions. We posit new functions for regulators of differentiation, the anti-viral response, and mitochondrial function during immune activation. By decomposing many high content measurements into the effects of perturbations, their interactions, and diverse cell metadata, Perturb-seq dramatically increases the scope of pooled genomic assays.

AB - Genetic screens help infer gene function in mammalian cells, but it has remained difficult to assay complex phenotypes—such as transcriptional profiles—at scale. Here, we develop Perturb-seq, combining single-cell RNA sequencing (RNA-seq) and clustered regularly interspaced short palindromic repeats (CRISPR)-based perturbations to perform many such assays in a pool. We demonstrate Perturb-seq by analyzing 200,000 cells in immune cells and cell lines, focusing on transcription factors regulating the response of dendritic cells to lipopolysaccharide (LPS). Perturb-seq accurately identifies individual gene targets, gene signatures, and cell states affected by individual perturbations and their genetic interactions. We posit new functions for regulators of differentiation, the anti-viral response, and mitochondrial function during immune activation. By decomposing many high content measurements into the effects of perturbations, their interactions, and diverse cell metadata, Perturb-seq dramatically increases the scope of pooled genomic assays.

KW - CRISPR

KW - epistasis

KW - genetic interactions

KW - pooled screen

KW - single-cell RNA-seq

UR - http://www.scopus.com/inward/record.url?scp=85006488344&partnerID=8YFLogxK

U2 - 10.1016/j.cell.2016.11.038

DO - 10.1016/j.cell.2016.11.038

M3 - Article

C2 - 27984732

AN - SCOPUS:85006488344

SN - 0092-8674

VL - 167

SP - 1853-1866.e17

JO - Cell

JF - Cell

IS - 7

ER -

Perturb-Seq: Dissecting Molecular Circuits with Scalable Single-Cell RNA Profiling of Pooled Genetic Screens

Abstract

Bibliographical note

Keywords

Access to Document

Other files and links

Fingerprint

Cite this