Enhanced immunogenicity of BCG through CRISPRi mediated depletion of AftC.

causes the disease tuberculosis and affects a third of the world's population. The recent COVID-19 pandemic exacerbated the situation with a projected 27% increase in tuberculosis related deaths. has an elaborate cell wall consisting of peptidoglycan, arabinogalactan and mycolic acids which shield the bacilli from the toxic bactericidal within phagocytes. Amongst, the numerous glycosyltransferase enzymes involved in mycobacterial cell wall biosynthesis, arabinofuranosyltransferase C () is responsible for the branching of the arabinan domain in both arabinogalactan and lipoarabinomannan. Using Clustered Regularly Interspaced Short Palindromic Repeats interference (CRISPRi) we have generated knockdowns in BCG and demonstrated the generation of a truncated, immunogenic lipoarabinomannan within its cell envelope. The depleted BCG mutants were unable to form characteristic mycobacterial pellicular biofilms and elicit a potent immunostimulatory phenotype compared to wild type BCG in a THP1 cell line. This study paves the way to further explore novel BCG mutants as promising vaccine boosters in preventing pulmonary tuberculosis.