1/31/2024 0 Comments Screen marker extensionBy integrating inducible CRISPRi/a machinery into this cell line, we developed a genetic screening system that enables robust knockdown and overexpression of endogenous genes in human microglia. These induced-transcription factor microglia-like cells (iTF-Microglia) resemble other iPSC-derived microglia 13, 14, 15, 16, 17, 18, 19 in their expression profiles, response to inflammatory stimuli, phagocytic capabilities and capacity to be cocultured with iPSC-derived neurons. To overcome these challenges, we developed a different approach for the generation of iPSC-derived microglia by generating a human iPSC line inducibly expressing six transcription factors that enable the generation of microglia-like cells in a rapid and efficient 8-day protocol. However, most existing protocols are lengthy and aim to recapitulate human microglia ontogeny 13, 14, 15, 16, 17, 18, 19, resulting in population bottlenecks during differentiation, which can skew the representation of the sgRNA library. This problem could be overcome by introducing sgRNAs at the iPSC stage. Pooled CRISPR screens rely on lentiviral transduction to introduce libraries of single guide RNAs (sgRNAs), but mature microglia are difficult to transduce with lentivirus. However, such screens have not previously been implemented in iPSC-derived microglia due to challenges inherent in available differentiation protocols. We recently provided a proof of principle for this strategy by establishing CRISPRi and CRISPRa platforms for genetic screens in iPSC-derived neurons 11, 12. When combined with induced pluripotent stem cell (iPSC) technology, they enable the investigation of cell-type-specific biology in human cells, including those derived from patients 10. Pooled CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) screens enable scalable modeling of changes in gene expression and genetic screens to uncover regulatory mechanisms. However, we do not systematically understand how these distinct microglial states contribute to brain function and disease, or the molecular mechanisms regulating these states.Ī promising approach to tackle these questions is enabled by CRISPR-based functional genomics in differentiated human cell types 10. To understand the molecular mechanisms underlying the role of microglia in disease and to target them therapeutically, it is necessary to bridge the gap between disease-associated genetic variants and changes in microglial function.Ī major challenge is that microglia adopt a large number of distinct functional states in health and disease, which are actively being mapped on the molecular level in mice and humans 3, 4, 5, 6, 7, 8, 9. Over the last decade, human genetics have pointed to a central role for microglia in brain diseases such as Alzheimer’s disease (AD) 2, where specific disease-associated genetic variants likely act in microglia, redefining them as potential drivers of AD. Microglia have a central role in brain development and homeostasis as well as in the pathogenesis of many brain disorders 1. Thus, our platform can systematically uncover regulators of microglial states, enabling their functional characterization and therapeutic targeting. A disease-associated state characterized by osteopontin (SPP1) expression was selectively depleted by colony-stimulating factor-1 (CSF1R) inhibition. A screen with single-cell RNA sequencing as the readout revealed that these microglia adopt a spectrum of states mirroring those observed in human brains and identified regulators of these states. These screens uncovered genes controlling microglia survival, activation and phagocytosis, including neurodegeneration-associated genes. We established inducible CRISPR interference and activation in this system and conducted three screens targeting the ‘druggable genome’. We developed an efficient 8-day protocol for the generation of microglia-like cells based on the inducible expression of six transcription factors. Here, we present a screening platform to systematically elucidate functional consequences of genetic perturbations in human induced pluripotent stem cell-derived microglia. However, we lack a systematic understanding of the underlying mechanisms. Microglia are emerging as key drivers of neurological diseases.
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