State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China; College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China.
State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China.
Genomics Proteomics Bioinformatics. 2022 Dec;20(6):1180-1196. doi: 10.1016/j.gpb.2021.01.008. Epub 2021 Dec 16.
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.
结核分枝杆菌是结核病（TB）的病原体，它仍然是全球单一传染病导致死亡的主要原因。新型抗结核药物和疫苗的开发受到结核分枝杆菌复杂且耗时的遗传操作技术的严重阻碍。在这里，我们利用结核分枝杆菌内源性的 III-A 型 CRISPR/Cas10 系统进行高效的基因编辑和 RNA 干扰（RNAi）。这种简单的方法只需要转化单个 mini-CRISPR 阵列质粒，从而避免了外源蛋白的引入和最小化了蛋白毒性。我们证明了该系统可以有效地和特异性地敲入/敲除结核分枝杆菌基因，这一结果通过 DNA 高通量测序得到了证实。该系统进一步应用于单基因和多基因 RNAi。此外，我们成功地进行了全基因组 RNAi 筛选，以鉴定调控结核分枝杆菌体外和细胞内生长的基因。该系统可以广泛用于探索结核分枝杆菌的功能基因组学，并有助于开发新型抗结核药物和疫苗。
