结核病（TB）是威胁人类健康的重要传染病，是全球十大死亡原因之一。耐药结核病治疗周期长、成功率低、治疗费用高。结核持留菌形成是其潜伏性感染、内原性复发和耐药性产生等一系列问题的重要原因之一。毒素－抗毒素 （Toxin-Antitoxin，TA）系统可能参与细菌应激的适应性反应，在细菌持留、药 物耐受中发挥着重要作用。结核分枝杆菌中有多达 90 多对 TA 基因，它们在结核分枝杆菌发病机理中的作用受到广泛关注，然而其在结核分枝杆菌中的生理功能尚不清楚。基于高通量测序的 CRISPR 文库筛选技术是功能基因组学的重要研究方法之一。本研究首先利用 CRISPR-Cas 系统辅助的非同源末端连接基因编辑技术 (CRISPR-NHEJ)在结核分枝杆菌中设计 90 个 sgRNA 靶向 47 个毒素基因进行基因敲除（knock-out）突变库的构建。利用该突变库在缺氧、酸性、氧化应激和饥饿等压力条件下进行筛选，我们发现一些毒素基因可能参与结核分枝杆菌在应激条件下的生长。本研究首次尝试在细菌中构建 CRISPR-KO 文库，为构建大型突变文库奠定了基础，并且提示 TA 可能在各种应激条件下发挥作用，为阐明 TA 在结核分枝杆菌生理中的作用奠定了基础。下一步计划对筛选到的基因进行验证。

Tuberculosis (TB) is an important infectious disease threatening human health and one of the top ten causes of death in the world. Drug-resistant tuberculosis has a long treatment cycle, low success rate and high treatment costs. The formation of mycobacteria persisters is one of the important reasons for its latent infection, long treatment cycle, endogenous relapse, and drug resistance. Toxin-Antitoxin (TA) systems may participate in the adaptive response of stresses and play an important role in the persistence and drug tolerance. There are as many as 90 pairs of TA genes in Mycobacterium tuberculosis genomes. The role of TA systems in the pathogenesis of M. tuberculosis has received wide attention, but their physiological functions are still unclear. CRISPR library screening technology based on high-throughput sequencing is one of the effective methods in functional genomics research. In this study, 90 sgRNAs were designed to target 47 toxin genes in M. tuberculosis using a CRISPR-Assisted Nonhomologous End-Joining Strategy （CRISPRNHEJ） to construct a knock-out gene mutation library. Using this library to screen under stress conditions such as hypoxia, acid, oxidative, nitrosative stress and starvation, we found that some toxin genes may be involved in the growth of M. tuberculosis under these conditions. This study is the first attempt of CRISPR-KO library screening in bacteria, laying a foundation for the construction of large mutant library and suggests the role of TA in the physiology of M. tuberculosis. The next step is to verify the phenotype of the selected genes.