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

• DOI: 10.7554/elife.60083
• Publication Date: 2020-11-05T14:00:47Z
• Authors: Timothy J de Wet; Kristy R Winkler; Musa Mhlanga; Valerie Mizrahi; Digby F Warner
• Keywords: 0301 basic medicine; QH301-705.5; Science; Mycobacterium smegmatis; 03 medical and health sciences; Drug Resistance, Bacterial; Image Processing, Computer-Assisted; Biology (General); Gene Library; 0303 health sciences; Q; R; CRISPRi; UMAP; Genetics and Genomics; Mycobacterium tuberculosis; Anti-Bacterial Agents; 3. Good health; Mycolic Acids; Genes, Bacterial; Gene Knockdown Techniques; Multigene Family; Medicine; CRISPR-Cas Systems; functional genomics; Genome, Bacterial; Metabolic Networks and Pathways; phenoprint

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.