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：
10518406
负责人：
DAVID S WEISS
金额：
$ 17.38万
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 2024-10-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10518406
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 Cessation of life Cloning Clostridium Clostridium difficile Communities 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 Future Gene Cluster Gene Expression Genes Genetic Transcription Guide RNA Health Hospitals Human Individual Infection Knowledge Lead Libraries Link Lipids Metabolism Methods Morphology Mutagenesis Nursing Homes Operon Organism Orthologous Gene Pathway interactions Persons Pharmaceutical Preparations Phase Phenotype Physiological Plasmids Process Protein Inhibition 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 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.

项目摘要在美国，梭状芽胞杆菌（梭状芽胞杆菌）艰难梭菌感染每年接近500,000人近30,000人死亡。疾病预防控制中心已宣布这种生物是对公共卫生的“紧急”威胁，是最高的威胁类别。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努力抗生素。尽管难以研究基本基因，但要操纵细菌基因的新技术表达正在克服这个障碍。一种用于研究细菌基因基因的新兴技术是称为CRISPR干扰（CRISPRI）。 CRISPRI使用短RNA引导一种称为DCAS9的蛋白质到基因的蛋白质，从而创建一个障碍，可防止RNA聚合酶转录该基因。 Crispri非常适合用于筛选大量基因，因为表达新目标只是克隆合成DNA的问题序列编码正确的导向RNA到质粒中，该过程很容易扩展。另一个关键功能我们的CRISPRI系统的是DCAS9是由木糖诱导的启动子表达的，这使我们能够控制基因表达的时间和程度。该提议的具体目标是部署Crispri 表征艰难梭菌中187个基本基因。在AIM 1中，我们将构建一个CRISPRI库并使用它审查我们目标列表中基因的本质。在AIM 2中，我们将确定基因敲低是否结果在形态缺陷中或增加对靶向多种细胞的抗生素的敏感性过程。这些分析的信息将使我们能够将许多基因分配给功能途径。它还将为开发新抗生素治疗艰难梭菌提供基本知识和工具感染。