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Cancer genomics studies have identified thousands of putative cancer driver genes(1). Development of high-throughput and accurate models to define the functions of these genes is a major challenge. Here we devised a scalable cancer-spheroid model and performed genome-wide CRISPR screens in 2D monolayers and 3D lung-cancer spheroids. CRISPR phenotypes in 3D more accurately recapitulated those of in vivo tumours, and genes with differential sensitivities between 2D and 3D conditions were highly enriched for genes that are mutated in lung cancers. These analyses also revealed drivers that are essential for cancer growth in 3D and in vivo, but not in 2D. Notably, we found that carboxypeptidase D is responsible for removal of a C-terminal RKRR motif(2) from the alpha-chain of the insulin-like growth factor 1 receptor that is critical for receptor activity. Carboxypeptidase D expression correlates with patient outcomes in patients with lung cancer, and loss of carboxypeptidase D reduced tumour growth. Our results reveal key differences between 2D and 3D cancer models, and establish a generalizable strategy for performing CRISPR screens in spheroids to reveal cancer vulnerabilities.

CRISPR screens in a 3D spheroid cancer model system more accurately recapitulate cancer phenotypes than existing 2D models and were used to identify carboxypeptidase D, acting via the IGF1R, as a 3D-specific driver of cancer growth.

Two epigenetic regulators-identified in an RNA interference screen in Caenhorhabditis elegans, and conserved in mammals-diminish mitochondrial function and accelerate the age-related deterioration of behaviour.

Multiple sclerosis is a chronic inflammatory disease of the CNS1. Astrocytes contribute to the pathogenesis of multiple sclerosis(2), but little is known about the heterogeneity of astrocytes and its regulation. Here we report the analysis of astrocytes in multiple sclerosis and its preclinical model experimental autoimmune encephalomyelitis (EAE) by single-cell RNA sequencing in combination with cell-specific Ribotag RNA profiling, assay for transposase-accessible chromatin with sequencing (ATAC-seq), chromatin immunoprecipitation with sequencing (ChIP-seq), genome-wide analysis of DNA methylation and in vivo CRISPR-Cas9-based genetic perturbations. We identified astrocytes in EAE and multiple sclerosis that were characterized by decreased expression of NRF2 and increased expression of MAFG, which cooperates with MAT2 alpha to promote DNA methylation and represses antioxidant and anti-inflammatory transcriptional programs. Granulocyte-macrophage colony-stimulating factor (GM-CSF) signalling in astrocytes drives the expression of MAFG and MAT2 alpha and pro-inflammatory transcriptional modules, contributing to CNS pathology in EAE and, potentially, multiple sclerosis. Our results identify candidate therapeutic targets in multiple sclerosis.

Single-cell RNA sequencing of cells from humans with multiple sclerosis and mice with a model of the disease identifies a population of disease-promoting astrocytes in which anti-oxidant and anti-inflammatory proteins are suppressed.

Single-cell RNA sequencing and spatial transcriptomics reveal that the somitogenesis clock is active in mouse gastruloids, which can be induced to generate somites with the correct rostral-caudal patterning.

Gastruloids are three-dimensional aggregates of embryonic stem cells that display key features of mammalian development after implantation, including germ-layer specification and axial organization(1-3). To date, the expression pattern of only a small number of genes in gastruloids has been explored with microscopy, and the extent to which genome-wide expression patterns in gastruloids mimic those in embryos is unclear. Here we compare mouse gastruloids with mouse embryos using single-cell RNA sequencing and spatial transcriptomics. We identify various embryonic cell types that were not previously known to be present in gastruloids, and show that key regulators of somitogenesis are expressed similarly between embryos and gastruloids. Using live imaging, we show that the somitogenesis clock is active in gastruloids and has dynamics that resemble those in vivo. Because gastruloids can be grown in large quantities, we performed a small screen that revealed how reduced FGF signalling induces a short-tail phenotype in embryos. Finally, we demonstrate that embedding in Matrigel induces gastruloids to generate somites with the correct rostral-caudal patterning, which appear sequentially in an anterior-to-posterior direction over time. This study thus shows the power of gastruloids as a model system for exploring development and somitogenesis in vitro in a high-throughput manner.