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

项目摘要 在美国，每年有近50万人感染艰难梭菌， 导致近3万人死亡疾病预防控制中心已宣布这种生物体对公共卫生构成“紧急”威胁， 威胁类别。迫切需要新的治疗方法。大多数抗生素抑制对生存力至关重要的蛋白质， 因此，更好地了解这些蛋白质和编码它们的基因可以促进开发新的 抗生素虽然必需基因很难研究，但操纵细菌基因的新技术 我们正在克服这一障碍。一种研究细菌必需基因的新兴技术是 CRISPR干扰（CRISPRi）CRISPRi使用一种短RNA来引导一种名为dCas9的蛋白质到达一个基因， 从而产生一个路障，阻止RNA聚合酶转录该基因。CRISPRi非常适合 因为抑制一个新的靶点仅仅是克隆一个合成的DNA 将正确的引导RNA编码序列插入质粒中，这是一个容易扩大规模的过程。另一个关键特性 我们的CRISPRi系统的一个优点是dCas9是从木糖诱导型启动子表达的，这使得我们能够控制 基因抑制的时间和程度该提案的具体目标是将CRISPRi部署到 鉴定了187个C.很难在目标1中，我们将构建CRISPRi文库并使用它 来检查我们目标清单上的基因的重要性在目标2中，我们将确定基因敲除是否会导致 或增加对一组抗生素的敏感性，所述抗生素被选择用于靶向多种细胞， 流程.从这些检测中获得的信息将使我们能够将这些基因中的许多基因分配给功能性的 途径。它还将为开发新的抗生素来治疗C。艰难 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.