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Rapid genetic screening in neurological disorders using CRISPR

Written by Liam Little, Science Writer

A recent article, published in Nature Neuroscience, aimed to understand the emerging roles of microglia, a category of cells found in the brain, as key drivers of neurological diseases. Using CRISPR interference and activation (CRISPRi/a), the scientists screened the effects of genetic perturbations in human induced pluripotent stem cell (iPSC)-derived microglia. The potential mechanisms of neurodegenerative-associated genes were uncovered, and sequencing showed that microglia mirror specific states observed in human brains.

A novel microglia model

Microglia play a central role in brain development and homeostasis. They are the main phagocytosing cells in the brain and are the first line of defence within the central nervous system. Genetic variants can turn microglia into pathogenic drivers of brain disorders, including Alzheimer’s disease. In this study, researchers, led by a team at the University of California, developed a novel 8-day protocol for the generation of induced-transcription factor microglia-like cells (iTF-Microglia) (Figure 1).

Figure 1: Differentiation of iPSCs into iTF-Microglia. Direct cell conversion was performed by over expressing transcription factors. By Day 8 iTF-Microglia were fully differentiated.

Pooled CRISPRi/a enables screening of gene expression to uncover regulatory mechanisms. The researchers integrated CRISPRi/a with the iTF-Microglia cells they generated to develop a robust and rapid genetic screening system for human microglia. The power of this platform was demonstrated with multiple large-scale genetic screens.

Determining the mechanisms of microglial biology

CRISPRi/a screening uncovered unique insights into microglia by exploring key aspects of their cell biology. Knockdown and overexpression of endogenous genes were performed targeting (1) microglial survival and proliferation, (2) modifiers of microglial activation and (3) modifiers of phagocytosis by microglia. Several genes that have previously been associated with neurodegenerative diseases were identified. The authors highlighted PFN1 and INPP5D as modulators of phagocytosis within microglia, indicating how genetic mutations in these genes can contribute to neurological diseases.

CRISPR droplet sequencing

The researchers sought more detailed insights into the genetic mechanisms that affect microglial functions. They used CRISPR droplet sequencing (CROP-seq), combining CRISPRi perturbation with single-cell RNA-sequencing (scRNA-seq) to analyse 39 genes of interest in over 58,000 iTF-Microglia (Figure 2).

Figure 2: CROP-seq workflow. Single-guide RNA (sgRNA) library introduced into iPSCs. iTF-Microglia differentiation and CRISPRi activity induced. scRNA-seq performed on iTF-Microglia to obtain single cell transcriptomes. TMP (trimethoprim), Dox (doxycycline).

CROP-seq revealed distinct iTF-Microglia transcriptional states, mirroring those observed in human brains. Different clusters were defined by differential expression of genes such as SPP1 and TP53. The authors highlighted SPP1, in particular, as this gene is upregulated in several disease-associated microglial states and expression can affect prognosis.

Considerations for future research

The authors expect this new platform to be applied to screen for other microglia-related phenotypes in the future. Areas where this platform could be used include using iPSCs from specific familial or sporadic diseases and performing co-cultures to observe how iTF-Microglia interact with other brain cell types. The authors also highlight the possibility of transplanting iTF-Microglia into mice to generate an in vivo diseased brain model.