Utilization of CRISPR interference to investigate the contribution of genes to pathogenesis in a macrophage model of Mycobacterium tuberculosis infection.

OBJECTIVES: There is an urgent need for novel drugs that target unique cellular pathways to combat infections caused by Mycobacterium tuberculosis. CRISPR interference (CRISPRi)-mediated transcriptional repression has recently been developed for use in mycobacteria as a genetic tool for identifying and validating essential genes as novel drug targets. Whilst CRISPRi has been applied to extracellular bacteria, no studies to date have determined whether CRISPRi can be used in M. tuberculosis infection models. METHODS: Using the human monocytic macrophage-like THP-1 cell line as a model for M. tuberculosis infection we investigated if CRISPRi can be activated within intracellular M. tuberculosis. RESULTS: The transcriptional repression of two candidate M. tuberculosis genes, i.e. mmpL3 and qcrB, leads to a reduction in viable M. tuberculosis within infected THP-1 cells. The reduction in viable colonies is dependent on both the level of CRISPRi-mediated repression and the duration of repression. CONCLUSIONS: These results highlight the utility of CRISPRi in exploring mycobacterial gene function and essentiality under a variety of conditions pertinent to host infection.