Development of a CRISPR interference system for mycobacteria

描述（由申请人提供）：迫切需要开发用于治疗结核病的新药，以缩短治疗时间，从而使用更少的资源实现更高的完成率，并解决日益严重的耐药结核病问题。目前几乎所有的抗生素都针对必需基因或过程。虽然抑制对毒力重要的基因的新方法可能提供一种替代方法，但靶向必需基因可能仍然是开发新的M。结核病抑制剂然而，对功能未知或不完全理解的必需基因的表征是具有挑战性的，因为它们不能简单地被删除。在没有无效突变的情况下，受调控的条件基因表达可以提供信息，但在M中难以实现。结核病使用现有的工具。源自化脓性链球菌的成簇的、规则间隔的短回文重复（CRISPR）系统的技术，使用Cas9内切核酸酶与小RNA一起提供序列特异性，已迅速成为真核生物中基因编辑的首选方法。该系统的最近变体，其中Cas9中的氨基酸取代导致缺乏核酸酶活性但保留了核酸酶活性的蛋白质。 RNA和DNA结合（dCas 9）已用于获得细菌和真核生物中基因表达的特异性抑制。这个小型RO 3项目的目标是使该系统适用于分枝杆菌，特别是M。结核这一目标将通过两个具体目标实现。首先，我们将构建E。大肠杆菌-分枝杆菌穿梭载体，其整合了无水四环素诱导的基因表达以表达dCas 9和所需的小RNA构建体，以靶向基因进行抑制。其次，我们将测试该系统，使用1）表达荧光mCherry蛋白的基因，以便可以容易地量化阻遏，以及2）两个天然染色体基因座，以便我们可以在使用该系统的条件下检查该系统的功能。通过靶向这些基因中的不同位点，我们将能够评估不同靶结合位点（例如启动子区与编码序列）导致目的基因阻遏的程度。一旦开发出来，该系统将有助于研究人员表征功能未知的必需基因，并将促进靶向这些基因的蛋白质产物的抑制剂的开发。

DESCRIPTION (provided by applicant): There is an urgent need to develop new drugs for the treatment of tuberculosis, both to shorten therapy so that higher completion rates can be achieved using fewer resources, and to address the growing problem of drug-resistant tuberculosis. Nearly all current antibiotics target essential genes or processes. Though new approaches that inhibit genes important for virulence may provide an alternative, targeting essential genes is likely to remain the predominant approach to develop new M. tuberculosis inhibitors. Characterization of essential genes whose function is unknown or incompletely understood, however, is challenging because they cannot simply be deleted. In the absence of null mutations, regulated conditional gene expression can be informative, but is difficult to achieve in M. tuberculosis using current tools. Technology derived from the clustered, regularly interspaced, short palindromic repeat (CRISPR) system of Streptococcus pyogenes, using the Cas9 endonuclease together with small RNAs to provide sequence specificity, has rapidly become a method of choice for gene editing in eukaryotes. A recent variation of this system, in which amino acid substitutions in Cas9 result in a protein that lacks nuclease activity but retains RNA and DNA binding (dCas9), has been used to obtain specific repression of gene expression in both bacteria and eukaryotes. The goal of this small RO3 project is to adapt this system for use in mycobacteria, particularly in M. tuberculosis. This goal will be achieved through two specific aims. First we will construct E. coli-mycobacterial shuttle vectors that incorporate anyhydrotetracycline-inducible gene expression to express dCas9 and the required small RNA constructs, to target genes for repression. Second we will test this system, using 1) a gene that expresses the fluorescent mCherry protein, so that repression can be readily quantified, and 2) two native chromosomal loci so that we can examine the function of this system under conditions in which it would be used. By targeting different sites in these genes we will be able to assess the extent to which different target binding sites, e.g. promoter region versus coding sequence, leads to repression of the gene of interest. Once developed, this system will be useful for investigators to characterize essential genes whose function is not known, and will facilitate the development of inhibitors that target the protein products of these genes.