成簇的规律间隔的短回文重复序列干扰(clustered regularly interspaced short palindromic repeat interference，CRISPRi)是一种新型转录抑制技术，该系统包含RNA介导的DNA内切酶dCas9和针对目的基因的特异性单向导RNA(single guide RNA，sgRNA)，通过形成DNA识别复合物特异性识别相应DNA序列以抑制目的基因的转录。异柠檬酸脱氢酶(isocitrate dehydrogenase，ICD)是三羧酸循环中的关键代谢酶，在分枝杆菌的碳代谢过程中发挥重要作用。本研究利用CRISPRi高效抑制分枝杆菌特定基因表达的方法构建耻垢分枝杆菌icd敲低(icd knockdown，ICD-KD)株。定量聚合酶链反应(quantitative polymerase chain reaction，qPCR)和蛋白免疫印迹检测结果显示，耻垢分枝杆菌中icd转录水平与ICD蛋白表达水平显著下降，表明采用CRISPRi技术成功构建了耻垢分枝杆菌ICD-KD株。进一步研究ICD-KD株的生长情况，测定其在固体培养基点板及液体培养基中的生长曲线，结果均显示ICD-KD株生长速率明显减慢，同时菌体内ICD酶活显著降低，提示ICD对分枝杆菌的生长存活起重要作用。本研究使用CRISPRi技术快速构建了分枝杆菌必需基因的敲低菌株，为后续研究分枝杆菌ICD在碳源代谢通路中的功能和碳通量流向调控机制提供了重要基础。

Clustered regularly interspaced short palindromic repeat interference (CRISPRi) is a new type of transcriptional repression technology. The system contains RNA-mediated DNA endonuclease dCas9 and specific single guide RNA (sgRNA) for the target gene. The DNA recognition complex recognizes the corresponding DNA sequence to suppress transcription of the target gene. Isocitrate dehydrogenase (ICD) is a key metabolic enzyme in the tricarboxylic acid cycle (TAC) and plays an important role in the carbon metabolism of Mycobacteria. In this study, icd knockdown (ICD-KD) strain of Mycobacterium smegmatis (M. smegmatis) was constructed by using CRISPRi to efficiently inhibit the expression of mycobacterial specific genes. The results of quantitative polymerase chain reaction (qPCR) and Western blotting showed the significant decrease in the transcriptional level and protein expression level of ICD, indicating that M. smegmatis ICD-KD strain was constructed successfully by using CRISPRi technique. The growth rate of ICD-KD strain decreased significantly. At the same time, enzyme activity of ICD decreased significantly. It is suggested that CRISPRi technology could rapidly construct knockdown strains of essential mycobacterial genes, laying an important foundation for the subsequent study of mycobacterial ICD function in the carbon source metabolic pathway and carbon flux flow in the TAC.