Reprogramming Mycobacterium tuberculosis CRISPR System for Gene Editing and Genome- wide RNA Interference Screening

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 con-firmed 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.

Automated summary

In this study, the endogenous type III-A CRISPR/Cas10 system of M. tuberculosis was utilized for efficient gene editing and RNA interference. It was proven that this system can successfully and specifically knock in/out M. tuberculosis genes as confirmed by DNA high-throughput sequencing. Furthermore, genome-wide RNA interference screening was performed to identify M. tuberculosis genes regulating in vitro and intracellular growth. This system can be widely used for exploring the functional genomics of M. tuberculosis and facilitating the development of novel anti-TB drugs and vaccines.