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Arrayed CRISPRi and quantitative imaging describe the morphotypic landscape of essential mycobacterial genes

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dc.contributor.authorde Wet, T.J.
dc.contributor.authorWinkler, K.R.
dc.contributor.authorMhlanga, M.
dc.contributor.authorMizrahi, V.
dc.contributor.authorWarner, D.F.
dc.contributor.departmentTimothy J de Wet, Kristy R Winkler, Musa Mhlanga, Valerie Mizrahi, Digby F Warner: SAMRC/NHLS/UCT Molecular Mycobacteriology Research Unit, Department of Pathology, University of Cape Town, Cape Town, South Africaen_US
dc.date.accessioned2024-03-22T19:56:10Z
dc.date.available2024-03-22T19:56:10Z
dc.date.epub2020
dc.date.issued2020-11-06
dc.description.abstractMycobacterium 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.en_US
dc.description.sponsorshipThis work was supported by grants from the US National Institute of Child Health and Human Devel- opment (NICHD) U01HD085531 (to DFW), the Research Council of Norway (R and D Project 261669 ‘Reversing antimicrobial resistance’) (to DFW), the South African Medical Research Council (SAMRC) (to VM), the National Research Foundation of South Africa (to VM and DFW), and core funding from the Wellcome Trust [203135/Z/16/Z]. Research reported in this publication was supported by the Strategic Health Innovation Partnerships (SHIP) Unit of the SAMRC with funds received from the South African Department of Science Innovation. TdW received partial funding support from the SAMRC through its Division of Research Capacity Development under the National Medical Students Research Training Programme, from funding received from the South African National Treasury. We are thankful to Jeremy Rock and Sarah Fortune for the CRISPRi vectors, Keith Derbyshire and Todd Gray for the Dendra-tagged translational fusions, Bree Aldridge and Trever Smith for useful discussions, and Mandy Mason and other members of the MMRU for constructive critique and technical advice throughout this project.en_US
dc.identifier.citationde 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.en_US
dc.identifier.doi10.7554/eLife.60083
dc.identifier.journalElifeen_US
dc.identifier.urihttps://pubmed.ncbi.nlm.nih.gov/33155979/
dc.identifier.urihttps://hdl.handle.net/11288/595672
dc.language.isoenen_US
dc.publisherElife Sciencesen_US
dc.research.unitMolecular Mycobacteriologyen_US
dc.rightsAttribution 3.0 United States*
dc.rights.urihttp://creativecommons.org/licenses/by/3.0/us/*
dc.subjectCRISPRien_US
dc.subjectMycobacterium tuberculosisen_US
dc.subjectUMAPen_US
dc.subjectGeneticsen_US
dc.subjectFunctional genomicsen_US
dc.subjectGenomicsen_US
dc.subjectInfectious diseaseen_US
dc.subjectSDG-03 Good health and well-beingen_US
dc.titleArrayed CRISPRi and quantitative imaging describe the morphotypic landscape of essential mycobacterial genesen_US
dc.typeArticleen_US
dspace.entity.typePublication
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