Arrayed CRISPRi and Quantitative Imaging Describe the Morphotypic Landscape of Essential Mycobacterial Genes
Arrayed CRISPRi and Quantitative Imaging Describe the Morphotypic Landscape of Essential Mycobacterial Genes
ABSTRACT Mycobacterium tuberculosis possesses a large number of genes of unknown or merely predicted function, undermining fundamental understanding of pathogenicity and drug susceptibility. To address this challenge, we developed a high-throughput functional genomics approach combining inducible CRISPR-interference and image-based analyses of morphological features and sub-cellular molecular localizations in the related non-pathogen, M. smegmatis. Applying automated imaging and analysis to an arrayed library of 272 essential gene knockdown mutants, we derive robust, quantitative descriptions of bacillary morphologies consequent on gene silencing. Leveraging statistical-learning, we demonstrate that functionally related genes cluster by morphotypic similarity and that this information can be used to infer gene function. Exploiting this observation, we reveal a previously unknown restriction-modification system, and identify filamentation as a defining mycobacterial response to histidine starvation. Our results support the application of large-scale image-based analyses for mycobacterial functional genomics, simultaneously establishing the utility of this approach for drug mechanism-of-action studies.
Mhlanga Musa M.、de Wet Timothy J.、Mizrahi Valerie、Warner Digby F.、Winkler Kristin R.
Institute of Infectious Disease and Molecular Medicine, University of Cape Town||Department of Integrative Biomedical Sciences, University of Cape TownSAMRC/NHLS/UCT Molecular Mycobacteriology Research Unit, Department of Pathology, University of Cape Town||Institute of Infectious Disease and Molecular Medicine, University of Cape TownSAMRC/NHLS/UCT Molecular Mycobacteriology Research Unit, Department of Pathology, University of Cape Town||Institute of Infectious Disease and Molecular Medicine, University of Cape Town||Wellcome Centre for Infectious Diseases Research in Africa, University of Cape TownSAMRC/NHLS/UCT Molecular Mycobacteriology Research Unit, Department of Pathology, University of Cape Town||Institute of Infectious Disease and Molecular Medicine, University of Cape Town||Wellcome Centre for Infectious Diseases Research in Africa, University of Cape TownSAMRC/NHLS/UCT Molecular Mycobacteriology Research Unit, Department of Pathology, University of Cape Town||Institute of Infectious Disease and Molecular Medicine, University of Cape Town
微生物学分子生物学生物科学研究方法、生物科学研究技术
Mhlanga Musa M.,de Wet Timothy J.,Mizrahi Valerie,Warner Digby F.,Winkler Kristin R..Arrayed CRISPRi and Quantitative Imaging Describe the Morphotypic Landscape of Essential Mycobacterial Genes[EB/OL].(2025-03-28)[2025-07-20].https://www.biorxiv.org/content/10.1101/2020.03.20.000372.点此复制
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