Screening for Virulence Genes in Mycobacterium tuberculosis Eukaryotic-like Secreted Proteins via Macrophage Screening Using the CRISPR Platform

Mycobacterium tuberculosis (M.tb), the causative agent of tuberculosis (TB), employs eukaryotic-like secretory proteins to subvert host immunity for intracellular survival. Using CRISPR-NHEJ genome editing, we constructed a secretory protein mutant library and identified Rv0066c and Rv3139 as critical determinants of M.tb survival in macrophages. Validation with knockout strains confirmed their essential roles, establishing these effectors as promising therapeutic targets for TB control.