CRISPR interference-enabled phenotyping of essential genes in C. difficile to aid in discovery of antibiotic targets

项目摘要 在美国，艰难梭菌感染每年造成近50万人死亡，居首位 造成近3万人死亡。美国疾病控制与预防中心宣布这种微生物是对公共健康的“紧急”威胁，这是最高的 威胁类别。迫切需要新的治疗方法。大多数抗生素会抑制对生存至关重要的蛋白质， 因此，更好地了解这些蛋白质和编码它们的基因可以推动开发新的 抗生素。尽管基本基因很难研究，但操纵细菌基因的新技术 表达正在克服这一障碍。研究细菌基本基因的一项新兴技术是 称为CRISPR干扰(CRISPRi)。CRISPRi使用一种短RNA来引导一种名为dCas9的蛋白质进入基因， 从而造成了一个障碍，阻止了RNA聚合酶转录该基因。CRISPRi非常适合 用于筛选大组基因，因为抑制一个新的靶点只需克隆合成的DNA 将编码正确的引导RNA的序列插入到质粒中，这一过程很容易放大。另一个关键功能 我们CRISPRi系统的一个特点是，dCas9是由木糖诱导的启动子表达的，这使得我们能够控制 基因抑制的时间和程度。这项提议的具体目标是将CRISPRi部署到 艰难梭菌187个假定必需基因的特征。在目标1中，我们将构建一个CRISPRi库并使用它 来检查我们目标名单上的基因的重要性。在目标2中，我们将确定基因敲除结果 在形态上的缺陷或增加对一组被选择来针对各种细胞的抗生素的敏感性 流程。来自这些分析的信息将使我们能够将这些基因中的许多分配到功能上 小路。它还将为开发治疗艰难梭菌的新抗生素提供基础知识和工具。 感染。

Project Summary Clostridioides (Clostridium) difficile infections strike close to 500,000 people a year in the United States, leading to nearly 30,000 deaths. The CDC has declared this organism an “urgent” threat to public health, the highest threat category. New treatments are sorely needed. Most antibiotics inhibit proteins that are essential for viability, so a better understanding of these proteins and the genes that encode them can advance efforts to develop new antibiotics. Although essential genes are difficult to study, new techniques for manipulating bacterial gene expression are overcoming this barrier. One emerging technology for studying essential genes in bacteria is called CRISPR interference (CRISPRi). CRISPRi uses a short RNA to guide a protein called dCas9 to a gene, thereby creating a roadblock that prevents RNA polymerase from transcribing that gene. CRISPRi is well-suited for screening large sets of genes because repressing a new target is simply a matter of cloning a synthetic DNA sequence encoding the right guide RNA into the plasmid, a process that is easily scaled-up. Another key feature of our CRISPRi system is that dCas9 is expressed from a xylose-inducible promoter, which allows us to control the timing and extent of gene repression. The specific objective of this proposal is to deploy CRISPRi to characterize 187 putatively essential genes in C. difficile. In Aim 1 we will construct a CRISPRi library and use it to vet the essentiality of the genes on our target list. In Aim 2 we will determine whether gene knockdown results in morphological defects or increases sensitivity to a panel of antibiotics chosen to target a variety of cellular processes. The information from these assays will enable us to assign many of these genes to functional pathways. It will also provide foundational knowledge and tools for developing new antibiotics to treat C. difficile infections.