The development of new therapeutic methods against M. tuberculosis infections can be drastically accelerated by facile, precise, and markless genetic manipulation methods that allow for rapid identification and characterization of new drug targets in M. tuberculosis. Current methods for genetic manipulation in M. tuberculosis include allelic exchange using long linear DNA fragments , specialized transduction based on mycobacteriophage , recombineering mediated by phage-encoded recombination system , oligonucleotide-mediated recombineering followed by Bxb1 integrase targeting and non-homologous end joining (NHEJ) induced by highly specific double strand break . These methods either use multiple transformation steps and thus require months to years for editing, or can only generate non-precise mutations and thus are not amenable for gene function exploration at a single-base resolution.