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Functional Characterisation of Active Enhancers in Human Astrocytes Using a High-throughput CRISPRi Screen

ABSTRACT: We used CROP-seq to perform a high-throughput, parallel screen of 979 candidate enhancer perturbations in normal human astrocytes (NHAs), a primary cell-line. We identified the target gene(s) for 145 of these candidates, which were enriched for transcription with eRNA, transcription factor footprinting, and superenhancer annotation. Most regulatory interactions were <50kb, targeting the nearest gene in ~50% of cases, but were not typically captured by eQTL or in silico predictions. These data elucidate the regulatory network of an understudied-but-crucial brain cell-type.

ORGANISM(S): Homo sapiens

PROVIDER: GSE236057 | GEO | 2025/01/01

REPOSITORIES: GEO

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Project description:Conditionally essential (CE) genes are required by pathogenic bacteria to establish and maintain infections. CE genes encode virulence factors, such as secretion systems and effector proteins, as well as biosynthetic enzymes that produce metabolites not found in the host environment. Due to their outsized importance in pathogenesis, CE gene products are attractive targets for the next generation of antimicrobials. However, the precise manipulation of CE gene expression in the context of infection is technically challenging, limiting our ability to understand the roles of CE genes in pathogenesis and accordingly design effective inhibitors. We previously developed a suite of CRISPR interference-based gene knockdown tools that are transferred by conjugation and stably integrate into bacterial genomes that we call Mobile-CRISPRi. Here, we show the efficacy of Mobile-CRISPRi in controlling CE gene expression in an animal infection model. We optimize Mobile-CRISPRi in Pseudomonas aeruginosa for use in a murine model of pneumonia by tuning the expression of CRISPRi components to avoid nonspecific toxicity. As a proof of principle, we demonstrate that knock down of a CE gene encoding the type III secretion system (T3SS) activator ExsA blocks effector protein secretion in culture and attenuates virulence in mice. We anticipate that Mobile-CRISPRi will be a valuable tool to probe the function of CE genes across many bacterial species and pathogenesis models.IMPORTANCE Antibiotic resistance is a growing threat to global health. To optimize the use of our existing antibiotics and identify new targets for future inhibitors, understanding the fundamental drivers of bacterial growth in the context of the host immune response is paramount. Historically, these genetic drivers have been difficult to manipulate precisely, as they are requisite for pathogen survival. Here, we provide the first application of Mobile-CRISPRi to study conditionally essential virulence genes in mouse models of lung infection through partial gene perturbation. We envision the use of Mobile-CRISPRi in future pathogenesis models and antibiotic target discovery efforts.

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Functional Characterisation of Active Enhancers in Human Astrocytes Using a High-throughput CRISPRi Screen

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