结核分枝杆菌遗传操作技术经过不断的发展和完善，其效率和应用范围都得到了很大提高，然而目前这些方法尚不能有效对结核菌基因尤其是必需基因进行原位的无痕遗传操作，这严重制约了对结核菌致病和耐药机制的解析。我们在以前的研究中，将CRISPR技术与噬菌体重组蛋白介导的重组技术联用，构建了可以在耻垢分枝杆菌中进行定点、高效基因组编辑的遗传操作工具，然而此技术却无法应用于结核菌。基于对CRISPR介导的重组技术的关键环节和结核菌遗传修复特点的深入分析，本研究拟通过提高结核分枝杆菌中的重组效率和从时间、剂量维度上精确调控CRISPR-Cas系统这两方面来实现该技术在结核菌中的应用。此外，我们将利用该技术对临床研究中发现的异烟肼耐药相关基因进行分析，探索INH耐药发展过程中联合突变的分子机制。本研究不仅为结核菌的生物学研究提供技术支持，而且为开发新型的耐药分子诊断技术提供理论依据。

Rcently the genetic manipulation methods of Mycobacterium tuberculosis have been greatly improved in the efficiency and application. However, these methods are inefficient for scarless and site-directed mutagenesis in chromosomal genes of M. tuberculosis, especially in essential genes, which hamper the discovery and validation of uncharacterized genes and pathways responsible for bacterial virulence and drug resistance. We have recently created a highly efficient system for marker-free and scarless genome editing in Mycobacterium smegmatis by combining CRISPR-Cas9 and recombineering. However, this system can not be applied to M. tuberculosis. Based on the analysis of the characteristics of CRISPR-assissted recombineering and M. tuberculosis DNA repair pathway, we will increase the recombination efficiency and control CRISPR-Cas9 clevage precisely across multiple dimensions, including those of dose and time, to allow this method to be extended to M. tuberculosis. In addition we will applied this method to investigate the roles of mutation of INH resistance-related genes and unveiling the mechanisam by which combined gene mutations function together to increase INH resistance. Our studies will not only facilitate the study of M. tuberculosis biology but also provide an insight on novel molecular diagnostic techniques.