Molecular Medicine Israel

A CRISPRi/a platform in human iPSC-derived microglia uncovers regulators of disease states

Abstract

Microglia are emerging as key drivers of neurological diseases. However, we lack a systematic understanding of the underlying mechanisms. Here, we present a screening platform to systematically elucidate functional consequences of genetic perturbations in human induced pluripotent stem cell-derived microglia. We developed an efficient 8-day protocol for the generation of microglia-like cells based on the inducible expression of six transcription factors. We established inducible CRISPR interference and activation in this system and conducted three screens targeting the ‘druggable genome’. These screens uncovered genes controlling microglia survival, activation and phagocytosis, including neurodegeneration-associated genes. 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. A disease-associated state characterized by osteopontin (SPP1) expression was selectively depleted by colony-stimulating factor-1 (CSF1R) inhibition. Thus, our platform can systematically uncover regulators of microglial states, enabling their functional characterization and therapeutic targeting.

Main

Microglia have a central role in brain development and homeostasis as well as in the pathogenesis of many brain disorders1. 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. 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.

A 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 humans3,4,5,6,7,8,9. However, we do not systematically understand how these distinct microglial states contribute to brain function and disease, or the molecular mechanisms regulating these states.

A promising approach to tackle these questions is enabled by CRISPR-based functional genomics in differentiated human cell types10. Pooled CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) screens enable scalable modeling of changes in gene expression and genetic screens to uncover regulatory mechanisms. 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 patients10. We recently provided a proof of principle for this strategy by establishing CRISPRi and CRISPRa platforms for genetic screens in iPSC-derived neurons11,12. However, such screens have not previously been implemented in iPSC-derived microglia due to challenges inherent in available differentiation protocols. Pooled CRISPR screens rely on lentiviral transduction to introduce libraries of single guide RNAs (sgRNAs), but mature microglia are difficult to transduce with lentivirus. This problem could be overcome by introducing sgRNAs at the iPSC stage. However, most existing protocols are lengthy and aim to recapitulate human microglia ontogeny13,14,15,16,17,18,19, resulting in population bottlenecks during differentiation, which can skew the representation of the sgRNA library.

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. These induced-transcription factor microglia-like cells (iTF-Microglia) resemble other iPSC-derived microglia13,14,15,16,17,18,19 in their expression profiles, response to inflammatory stimuli, phagocytic capabilities and capacity to be cocultured with iPSC-derived neurons. By 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. Using this platform, we conducted pooled CRISPRi and CRISPRa screens for modifiers of survival, phagocytosis and inflammatory activation, which uncovered microglia-specific genes controlling these phenotypes. A screen with single-cell RNA sequencing (scRNA-seq) as the readout revealed that these microglia adopt a spectrum of states mirroring those observed in human brains, and pinpointed regulators of specific states, which can enable the functional characterization and therapeutic targeting of these states.

Results

Rapid and scalable production of microglia-like cells

We set out to create a fast, robust and scalable differentiation protocol to differentiate iPSCs to microglia-like cells for use in CRISPR screens. To this end, we developed a strategy based on direct cell fate conversion by overexpression of transcription factors. Based on transcriptomic and developmental data20,21,22, we selected six transcription factors highly expressed in human microglia: Hematopoietic Transcription Factor PU.1, MAF BZIP Transcription Factor B (MAFB), CCAAT Enhancer Binding Protein Alpha (CEBPα), CCAAT Enhancer Binding Protein Beta (CEBPβ), Interferon Regulatory Factor 5 (IRF5) and Interferon Regulatory Factor 8 (IRF8). We engineered an iPSC line with two integrated cassettes for the doxycycline-inducible expression of three transcription factors each in the Citrate Lyase Beta Like (CLYBL) and Adeno-Associated Virus Integration Site 1 (AAVS1) safe-harbor loci (Fig. 1a).

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