SUMMARY 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-seq and 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.

Summary The recent discovery that normal and neoplastic epithelial cells re-enter the stem-cell state raised an intriguing possibility in the context of cancer pathogenesis: the aggressiveness of carcinomas derives not from their existing content of cancer stem cells (CSCs), but from their proclivity to generate new CSCs from non-CSC populations. Here we demonstrate that non-CSCs of human basal breast cancers are plastic cell populations that readily switch from a non-CSC to CSC-state. The observed cell plasticity is dependent on ZEB1, a key regulator of the epithelial-mesenchymal transition. We find plastic non-CSCs maintain the ZEB1 promoter in a bivalent chromatin configuration enabling them to respond readily to microenvironmental signals, such as TGFbeta. In response, the ZEB1 promoter converts from a bivalent to active chromatin configuration, ZEB1 transcription increases and non-CSCs subsequently enter the CSC state. Our findings support a dynamic model where interconversions between low and high tumorigenic states occur frequently, thereby increasing tumorigenic and malignant potential.

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Expression of co-inhibitory receptors, such as CTLA-4 and PD-1, on effector T cells is a key mechanism for ensuring immune homeostasis. Dysregulated co-inhibitory receptor expression on CD4+ T cells promotes autoimmunity while sustained overexpression on CD8+ T cells promotes T cell dysfunction or exhaustion, leading to impaired ability to clear chronic viral infections and cancer1,2. Here, we used RNA and protein expression profiling at single-cell resolution to identify a module of co-inhibitory receptors that includes not only several known co-inhibitory receptors (PD-1, Tim-3, Lag-3, and TIGIT), but also a number of novel surface receptors. We functionally validated two novel co-inhibitory receptors, Activated protein C receptor (Procr) and Podoplanin (Pdpn). The module of co-inhibitory receptors is co-expressed in both CD4+ and CD8+ T cells and is part of a larger co-inhibitory gene program that is shared by non-responsive T cells in multiple physiological contexts and is driven by the immunoregulatory cytokine IL-27. Computational analysis identified the transcription factors Prdm1 and c-Maf as cooperative regulators of the co-inhibitory module, which we validated experimentally. This molecular circuit underlies the co-expression of co-inhibitory receptors in T cells and identifies novel regulators of T cell function with the potential to regulate autoimmunity and tumor immunity.