Mycobacterium tuberculosisis the causative agent of tuberculosis (TB), which is still the leading cause of mortality from a single infectious disease worldwide. The development of novel anti-TB drugs and vaccines is severely hampered by the complicated and time-consuming genetic manipulation techniques forM. tuberculosis.Here, we harnessed an endogenous type III-A CRISPR/Cas10 system ofM. tuberculosisfor efficientgene editingand RNA interference (RNAi). This simple and easy method only needs to transform a single mini-CRISPR array plasmid, thus avoiding the introduction of exogenous protein and minimizing proteotoxicity. We demonstrated thatM. tuberculosisgenes can be efficiently and specifically knocked in/out by this system as confirmed by DNA high-throughput sequencing. This system was further applied to single- and multiple-gene RNAi. Moreover, we successfully performedgenome-wide RNAi screeningto identifyM. tuberculosisgenes regulatingin vitroand intracellular growth. This system can be extensively used for exploring the functional genomics ofM. tuberculosisand facilitate the development of novel anti-TB drugs and vaccines.