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

Cell. 2016 Dec 15;167(7):1853-1866.e17. doi: 10.1016/j.cell.2016.11.038.

Authors

Affiliations

¹ Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Harvard-MIT Division of Health Sciences and Technology, Cambridge, MA 02139, USA.
² Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA.
³ Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Department of Systems Biology, Harvard Medical School, Boston, MA 02115, USA.
⁴ Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Computational and Systems Biology, Massachusetts Institute of Technology, Cambridge, MA 02140, USA.
⁵ Department of Cellular and Molecular Pharmacology, California Institute of Quantitative Biosciences, Center for RNA Systems Biology, University of California, San Francisco, San Francisco, CA 94158, USA.
⁶ Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Department of Systems Biology, Harvard Medical School, Boston, MA 02115, USA; Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02140, USA.
⁷ Department of Cellular and Molecular Pharmacology, California Institute of Quantitative Biosciences, Center for RNA Systems Biology, University of California, San Francisco, San Francisco, CA 94158, USA; Howard Hughes Medical Institute, Chevy Chase, MD 20815, USA.
⁸ Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; School of Engineering and Computer Science and Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem 91904, Israel.
⁹ Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02140, USA; Howard Hughes Medical Institute, Chevy Chase, MD 20815, USA. Electronic address: aregev@broadinstitute.org.

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.

MeSH terms

Animals
Cell Cycle
Clustered Regularly Interspaced Short Palindromic Repeats
Feedback
Gene Expression Profiling
Gene Knockdown Techniques
Humans
K562 Cells
Mice
Mice, Transgenic
Sequence Analysis, RNA / methods*
Single-Cell Analysis / methods*
Transcription Factors / metabolism

Substances

Transcription Factors

Abstract

MeSH terms

Substances

Grants and funding