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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.

项目总结