OR33

1:Research Institute for Medicines, Faculdade de Farmácia, Universidade de Lisboa, Lisboa, Portugal; 2:Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, United Kingdom; 3:Global Health and Tropical Medicine, GHTM, LA-REAL, Instituto de Higiene e Medicina Tropical, IHMT, Universidade NOVA de Lisboa, Lisboa, Portugal

The current antituberculous drug portfolio remains insufficient in effectively treating drug-resistant tuberculosis (TB). The inclusion of the latest approved drugs bedaquiline, pretomanid and delamanid has already contributed to the emergence of drug resistance mutations capable of rendering these antibiotics ineffective. Hence, expanding the antituberculous drug repertoire is paramount and new strategies are required to accelerate the drug discovery process. As such, a strategy was pursued to identify highly conserved genes in Mycobacterium tuberculosis that could serve as potential drug targets. A genome-wide analysis of 12315 clinical strains was performed to investigate the evolutionary dynamics of 4039 mycobacterial genes and predict which genes are under strong purifying selection. Combined with a genomic subtractive workflow, a small sample of genes was selected to further validate their essentiality in vitro through CRISPR interference-mediated gene repression. Thirteen genes were selected for having high rates of sites under purifying selection of up to 18% and non-synonymous/synonymous substitution rates ratios as low as 0.07, through statistical inference methodologies and evolutionary models. Most of these genes are responsible for regulating important mycobacterial biosynthetic pathways, such as mycolic acid synthesis, protein synthesis, gene expression, cell division, host-pathogen interactions, and modulating the host immune response. For each gene, recombinant strains of M. tuberculosis holding a sequence-specific version of the CRISPR/dCas9 system were produced, gene repression was induced and bacterial viability was measured.  Downregulation of approximately 85% of the tested genes led to a complete loss of viability and were therefore considered essential for mycobacterial survival.