To address these challenges in mycobacteria gene editing, we developed highly efficient PAM-expanded St1Cas9 C-to-T and C-to-G base editors for programmed base editing in mycobacteria. These systems can achieve precise single base substitutions by a single transformation step, thereby substantially reducing the time and efforts for genetic manipulation. Moreover, the expression of the operonic genes downstream of the Cas protein binding sites that can be silenced by the CRISPRi system , will not be affected by the base editing systems. In addition, the base editing systems are amenable for highly efficient multiplexed editing, which are extremely difficult to achieve using serial editing that is prohibitively time consuming in slow-growing pathogens. Because only a 20 nt spacer sequence is required for targeting, in addition to single gene editing, the systems can be further engineered to be a high-throughput gene knockout screening method, which would allow for the systematical discovery of new drug targets and facilitate new therapeutic method development in mycobacteria.