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

Tuberculosis (TB), caused by Mycobacterium tuberculosis (M.tb), remains a serious infectious disease posing significant global health challenges. A critical evolutionary feature of M.tb is its genome encoding a set of eukaryotic-like secreted proteins, which facilitate intracellular survival by manipulating host immune responses. In this study, we constructed a mutant library targeting eukaryotic-like secreted proteins using CRISPR-NHEJ genome editing. From this library, we identified genes essential for M.tb survival in THP-1 macrophages. Subsequent validation using large-fragment knockout strains confirmed that deletion of Rv0066c or Rv3139 substantially compromised M.tb intracellular persistence. Collectively, this work establishes a eukaryotic-like secretory protein mutant library and identifies two novel M.tb effectors governing intracellular survival, providing potential new targets for anti-TB drug development.