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

Highlights•Pooled CRISPR screen with scRNA-seq readout•Integrated model of perturbations, single cell phenotypes, and epistatic interactions•Effect of TFs on genes, programs, and states in LPS response in immune cells•Downsampling assessment of feasibility of genome-wide or combinatorial screensSummaryGenetic 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.Graphical abstract