Genetic manipulation is of vital importance in facilitating the study of M. tuberculosis biology and drug-target exploration. However, the highly desirable scarless, precise, and markless editing in M. tuberculosis relies on HDR and requires months to years for editing. CRISPR-assisted HDR methods have been developed for rapid and precise genome editing in a number of bacterial species . However, it is not applicable in M. tuberculosis likely due to the lack of a CRISPR-compatible HDR system.