de Wet, T.J.Winkler, K.R.Mhlanga, M.Mizrahi, V.Warner, D.F.2024-03-222024-03-222020-11-06de Wet TJ, Winkler KR, Mhlanga M, Mizrahi V, Warner DF. Arrayed CRISPRi and quantitative imaging describe the morphotypic landscape of essential mycobacterial genes. Elife. 2020 Nov 6;9:e60083. doi: 10.7554/eLife.60083.10.7554/eLife.60083https://pubmed.ncbi.nlm.nih.gov/33155979/https://hdl.handle.net/11288/595672Mycobacterium tuberculosis possesses a large number of genes of unknown or 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 chromosomal localizations in the related non-pathogen, M. smegmatis. Applying automated imaging and analysis to 263 essential gene knockdown mutants in an arrayed library, 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 inform investigations of gene function. Exploiting this observation, we infer the existence of a mycobacterial 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.enAttribution 3.0 United Stateshttp://creativecommons.org/licenses/by/3.0/us/CRISPRiMycobacterium tuberculosisUMAPGeneticsFunctional genomicsGenomicsInfectious diseaseSDG-03 Good health and well-beingArticleElife