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.
Bibliographical noteFunding 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We thank A. Basu, C. Muus, C. Rodman, and N. Rogel for help in unpublished pilot; D. Feldman for feedback; K. Shekhar for vector design; B. Cleary and A. Bloemendal for discussions on combinatorial models; T. Wang for an unpublished plasmid; L. Gaffney for help with figures; and the Genomics Platform for sequencing.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.
© 2016 Elsevier Inc.
- genetic interactions
- pooled screen
- single-cell RNA-seq