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

基本信息

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
项目类别：
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

项目摘要

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，导致疾病预防控制中心 (CDC) 已宣布这种微生物对公共健康构成“紧急”威胁，是最高的威胁。迫切需要新的治疗方法来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尽管必需基因很难研究，但是操纵细菌基因的新技术。研究细菌必需基因的一项新兴技术正在克服这一障碍。称为 CRISPR 干扰 (CRISPRi)，CRISPRi 使用短 RNA 引导称为 dCas9 的蛋白质到达基因，因此，创建一个阻止 RNA 聚合酶转录该基因的障碍是非常合适的。用于筛选大量基因，因为抑制新靶标只需克隆合成 DNA 将正确的引导 RNA 编码到质粒中的序列，这是一个易于放大的过程。我们的 CRISPRi 系统的一个特点是 dCas9 由木糖诱导型启动子表达，这使我们能够控制该提案的具体目标是将 CRISPRi 部署到基因抑制的时间和程度。表征艰难梭菌中 187 个假定的必需基因在目标 1 中，我们将构建一个 CRISPRi 文库并使用它。审查目标列表中基因的重要性，在目标 2 中，我们将确定是否会产生基因敲除结果。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。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。