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

对艰难梭菌中的必需基因进行 CRISPR 干扰表型分析，以帮助发现抗生素靶标

• 批准号: 10369416
• 负责人: DAVID S WEISS
• 金额: $ 20.86万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-11-02 至 2023-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10369416
• 关键词: Affect; Antibiotic Resistance; Antibiotics; Bacillus subtilis; Bacteria; Bacterial Genes; Bioinformatics; Biological Assay; CRISPR interference; Categories; Cell Wall; Cell division; Cell physiology; Cells; Centers for Disease Control and Prevention (U.S.); Cessation of life; Cloning; Clostridium difficile; Communities; Conflict (Psychology); Cytolysis; DNA Sequence; DNA biosynthesis; DNA-Directed RNA Polymerase; Defect; Development; Drug Interactions; Drug Targeting; Emerging Technologies; Escherichia coli; Essential Genes; Failure; Filament; Fluorescence Microscopy; Foundations; Future; Gene Cluster; Gene Expression; Genes; Genetic Transcription; Guide RNA; Health; Hospital Nursing; Human; Individual; Infection; Knowledge; Lead; Libraries; Light; Link; Lipids; Metabolism; Methods; Morphology; Mutagenesis; Nursing Homes; Operon; Organism; Orthologous Gene; Pathway interactions; Persons; Pharmaceutical Preparations; Phase; Phenotype; Physiological; Plasmids; Process; Proteins; Protocols documentation; Public Health; Quantitative Reverse Transcriptase PCR; RNA; Repression; Research; Research Personnel; Resources; Rod; Role; Shapes; System; Techniques; Translations; United States; Work; Xylose; base; deep sequencing; experimental study; follow-up; gene function; gene repression; glycosyltransferase; gut microbiota; improved; innovation; insight; interest; knock-down; microbiota; new therapeutic target; paralogous gene; prevent; promoter; protein-histidine kinase; repository; scale up; screening; segregation; synergism; synthetic construct; therapy outcome; tool; transposon sequencing

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.

项目概要 在美国，每年有近 50 万人被艰难梭菌 (Clostridium difficile) 感染，导致 导致近 30,000 人死亡。美国疾病控制与预防中心 (CDC) 宣布这种生物体对公共健康构成“紧急”威胁，是最高级别的威胁 威胁类别。迫切需要新的治疗方法。大多数抗生素会抑制生存所必需的蛋白质， 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蛋白质的努力 抗生素。尽管必需基因很难研究，但操纵细菌基因的新技术 表达正在克服这一障碍。研究细菌必需基因的一项新兴技术是 称为 CRISPR 干扰 (CRISPRi)。 CRISPRi 使用短 RNA 将称为 dCas9 的蛋白质引导至基因， 从而形成一个障碍，阻止 RNA 聚合酶转录该基因。 CRISPRi 非常适合 用于筛选大量基因，因为抑制新靶标只需克隆合成 DNA 将编码正确向导RNA的序列插入质粒中，这一过程很容易扩大规模。另一个关键功能 我们的 CRISPRi 系统的一个特点是 dCas9 由木糖诱导型启动子表达，这使我们能够控制 基因抑制的时间和程度。该提案的具体目标是将 CRISPRi 部署到 描述了艰难梭菌中 187 个推定必需基因的特征。在目标 1 中，我们将构建 CRISPRi 文库并使用它 审查我们目标列表上基因的重要性。在目标 2 中，我们将确定基因敲低是否会产生结果 形态缺陷或增加对一组针对多种细胞的抗生素的敏感性 流程。来自这些测定的信息将使我们能够将许多这些基因分配给功能性基因。 途径。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。