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 journalArticlepeer-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 languageAmerican English
Pages (from-to)1853-1866.e17
JournalCell
Volume167
Issue number7
DOIs
StatePublished - 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We thank A. Basu, C. Muus, C. Rodman, and N. Rogel for help in unpublished pilot; D. Feldman for feedback; K. Shekhar for vector design; B. Cleary and A. Bloemendal for discussions on combinatorial models; T. Wang for an unpublished plasmid; L. Gaffney for help with figures; and the Genomics Platform for sequencing.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

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