After transformation, all the colonies of M. smegmatis or M. tuberculosis were collected from the plates and the genomic DNAs were extracted using the Rapid Bacterial Genomic DNA Isolation Kit (Sangon Biotech, Shanghai, China). The target region was amplified with the Easy Taq DNA Polymerase (TransGen, Beijing, China) using specific primers for the target region. The PCR products were sent out for Sanger sequencing and the editing efficiency was calculated by using EditR . leuB or leuC knockout using CTBE evolve Spacers were designed and inserted into the CTBEevolve plasmid. The successfully constructed plasmids were electroporated into M. smegmatis mc 2 155 competent cells.