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Construction of a novel CRISPRi-based tool for silencing of multiple genes in Mycobacterium tuberculosis.
Plasmid ( IF 2.6 ) Pub Date : 2020-06-11 , DOI: 10.1016/j.plasmid.2020.102515
Nisheeth Agarwal 1
Affiliation  

Due to lipid-rich cell wall, slow growth and pathogenic nature, it is difficult to manipulate Mycobacterium tuberculosis (Mtb) genome by conventional tools. Recently we have introduced a novel CRISPRi approach for repression of genes in mycobacteria. Although the existing CRISPRi plasmid is proven useful for silencing individual targets, disruption of multiple ORFs remains challenging in mycobacteria. Herein, we report construction of the guide sequence expressing plasmid, pGrna to facilitate cloning and expression of multiple guide sequence cassettes targeting a versatile set of Mtb genes from a single plasmid. Using the modified plasmid, pGrna2, it was shown that expression of all the 10 extracellular sigma factor-encoding genes together with sigB and sigF can be efficiently repressed in Mtb expressing dCas9. In vitro growth analysis indicates that simultaneous knockdown of these non-essential transcriptional regulators is lethal for growth. Importantly, the Δ12sig strain exhibits sensitivity to transcriptional inhibitor rifampicin and oxidative stress diamide, further implying involvement of these genes in controlling bacterial stress response. To the best of my knowledge, this is the first report wherein 12 genes have been efficiently silenced together in a single recombinant strain of Mtb. The modified pGrna2 plasmid offers a powerful tool to decipher the functioning of genes that are redundant or regulate a particular metabolic pathway and can be useful in identification of novel anti-tuberculosis drug targets.



中文翻译:

构建一种基于CRISPRi的新型工具,用于沉默结核分枝杆菌中的多个基因。

由于富含脂质的细胞壁,缓慢的生长和致病性,难以通过常规工具操纵结核分枝杆菌(Mtb)基因组。最近,我们引入了一种新颖的CRISPRi方法来抑制分枝杆菌中的基因。尽管已证明现有的CRISPRi质粒可用于沉默单个靶标,但在分枝杆菌中破坏多个ORF仍然具有挑战性。在本文中,我们报道了指导序列表达质粒pGrna的构建,以促进克隆和表达针对单个质粒的一组多功能Mtb基因的多个指导序列盒。使用修饰的质粒pGrna2,显示了所有10个胞外sigma因子编码基因以及sigBsigF的表达可以在表达dCas9的Mtb中得到有效抑制。体外生长分析表明,同时敲低这些非必需的转录调节子对生长具有致命性。重要的是,Δ12sig菌株对转录抑制剂利福平和氧化应激二酰胺表现出敏感性,进一步暗示了这些基因参与控制细菌应激反应。据我所知,这是第一个报道,其中在单个Mtb重组菌株中有效地使12个基因一起沉默。修饰的pGrna2质粒为破译冗余基因或调节特定代谢途径的基因的功能提供了有力工具,可用于鉴定新型抗结核药物靶标。

更新日期:2020-06-11
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