Mycobacterium tuberculosis is 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 s...Mycobacterium tuberculosis is 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 for M. tuberculosis. Here, we harnessed an endogenous type Ⅲ-A CRISPR/Cas10 system of M. tuberculosis for efficient gene editing and 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 that M. tuberculosis genes 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 performed genome-wide RNAi screening to identify M. tuberculosis genes regulating in vitro and intracellular growth. This system can be extensively used for exploring the functional genomics of M. tuberculosis and facilitate the development of novel anti-TB drugs and vaccines.展开更多
基金supported by the National Key R&D Program of China(Grant No.2017YFD0500303)the National Natural Science Foundation of China(Grant Nos.C180501 and 31602061)+1 种基金the Huazhong Agricultural University Scientific&Technological Self-innovation Foundation,China(Grant Nos.2662017PY105 and 2662017PY105)the Doctoral Fund of Ministry of Education of China(Grant No.131012).
文摘Mycobacterium tuberculosis is 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 for M. tuberculosis. Here, we harnessed an endogenous type Ⅲ-A CRISPR/Cas10 system of M. tuberculosis for efficient gene editing and 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 that M. tuberculosis genes 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 performed genome-wide RNAi screening to identify M. tuberculosis genes regulating in vitro and intracellular growth. This system can be extensively used for exploring the functional genomics of M. tuberculosis and facilitate the development of novel anti-TB drugs and vaccines.
