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bioRxiv · 10.1101/2020.03.20.000372

Arrayed CRISPRi and Quantitative Imaging Describe the Morphotypic Landscape of Essential Mycobacterial Genes

de Wet, T. J.·Winkler, K. R.·Mhlanga, M. M.·Mizrahi, V.·Warner, D. F.
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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.

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de Wet, T. J., Winkler, K. R., Mhlanga, M. M., Mizrahi, V., Warner, D. F.. 2020-03-22. Arrayed CRISPRi and Quantitative Imaging Describe the Morphotypic Landscape of Essential Mycobacterial Genes. https://doi.org/10.1101/2020.03.20.000372

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