Abstract
Mycobacterium 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.
Highlights
The acquisition of genomic data continues to exceed significantly the pace at which corresponding functional information can be obtained (Kemble, Nghe et al 2019)
Leveraging available gene essentiality and CRISPRi guide efficacy data, we describe the construction of an arrayed collection of 272 validated CRISPRi mutants targeting essential M. smegmatis homologs of M. tuberculosis genes
We leveraged an optimized mycobacterial CRISPRi system (Rock, Hopkins et al 2017) and previously generated genome-wide CRISPRi-Seq data from which we were able to identify the set of highest efficiency single-guideRNAs targeting 294 essential M
Summary
The acquisition of genomic data continues to exceed significantly the pace at which corresponding functional information can be obtained (Kemble, Nghe et al 2019). This aphorism applies to Mycobacterium tuberculosis, etiological agent of tuberculosis (TB) and the leading cause of death globally from an infectious disease The most commonly applied functional genomics methods in bacteria – transposonsequencing (Tn-Seq) (Opijnen and Camilli 2013) and, increasingly, CRISPR-interference (CRISPRi)-Seq (de Wet, Gobe et al 2018, Wang, Guan et al 2018, Lee, Ostrov et al 2019) –
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