Development of a CRISPR interference system for mycobacteria

描述(申请人提供)：迫切需要开发治疗结核病的新药，既要缩短治疗时间，以便用更少的资源实现更高的完成率，又要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要的基因或过程。尽管抑制对毒力重要的基因的新方法可能会提供另一种选择，但靶向必要基因可能仍然是开发新的结核分枝杆菌抑制剂的主要方法。然而，对其功能未知或不完全了解的必要基因的表征是具有挑战性的，因为它们不能简单地被删除。在没有零突变的情况下，受调控的条件基因表达可以提供信息，但使用目前的工具很难在结核分枝杆菌中实现。起源于化脓性链球菌的簇状、规则间隔、短回文重复(CRISPR)系统的技术，利用Cas9内切酶和小RNA来提供序列特异性，已经迅速成为真核生物基因编辑的一种选择方法。这一系统的最新变体，其中Cas9中的氨基酸替换导致一种蛋白质缺乏核酸酶活性，但保留了 RNA和DNA结合(DCas9)，已经被用来获得细菌和真核生物中基因表达的特异性抑制。这个小型R03项目的目标是使这一系统适用于分枝杆菌，特别是结核分枝杆菌。这一目标将通过两个具体目标来实现。首先，我们将构建大肠杆菌-分枝杆菌穿梭载体，融合任何氢四环素诱导的基因表达，以表达dCas9和所需的小RNA结构，以靶向抑制基因。其次，我们将测试这个系统，使用1)一个表达荧光mCherry蛋白的基因，这样就可以很容易地量化抑制，以及2)两个天然染色体位置，这样我们就可以检查这个系统在使用条件下的功能。通过针对这些基因中的不同位点，我们将能够评估不同的靶结合位点，例如启动子区域和编码序列，导致目的基因抑制的程度。一旦开发出来，这个系统将有助于研究人员确定功能未知的必要基因的特征，并将促进针对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.