CRISPR and Virulence Core

CRISPR 和毒力核心

• 批准号: 10540799
• 负责人: Bridget Marie Barker
• 金额: $ 21.39万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-01-01 至 2026-12-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10540799
• 关键词: Affect; Algorithms; Antigens; Applied Genetics; Bioinformatics; Biological Assay; CRISPR/Cas technology; Candidate Disease Gene; Cell Culture Techniques; Cells; Centers for Disease Control and Prevention (U.S.); Clustered Regularly Interspaced Short Palindromic Repeats; Coccidioides; Coccidioides immitis; Coccidioides posadasii; Coccidioidomycosis; Coculture Techniques; Complex; Data; Data Analyses; Defect; Development; Diagnostic; Disease; Disease Outcome; Engineering; Fungal Drug Resistance; Gene Deletion; Gene Targeting; Genes; Genetic; Genetic Transcription; Goals; Human; Immune; In Vitro; Incidence; Infection; Label; Laboratories; Lead; Length of Stay; Macrophage; Marketing; Methodology; Modeling; Morphology; Mus; Pathogenesis; Pathogenicity; Pathway interactions; Patients; Pattern; Phase; Phenotype; Plasmids; Play; Proteins; Reporting; Research; Research Proposals; Role; Sister; Technology; Testing; Therapeutic; Time; Transcript; Vaccines; Virulence; Virulence Factors; Visualization software; Work; candidate identification; cost; desert fever; experience; experimental study; gene function; genetic approach; genome analysis; genome wide association study; improved; in vitro Assay; insight; interest; mouse model; mutant; novel; pathogen; pathogenic fungus; programs; promoter; reverse genetics; tool; trait; vaccine development

PROJECT SUMMARY/ABSTRACT Little is known about virulence mechanisms for most emerging fungal pathogens. Coccidioides immitis and C. posadasii, are of specific concern due to rapid increase in disease (coccidioidomycosis, aka Valley fever) incidence in the US, with a more than 10-fold increase in reported disease over the last 20 years. The Centers for Disease Control and Prevention (CDC) report that average cost for treating Valley fever is $50,000 per patient, including extensive hospital stays and treatment regimes. Use of CRISPR/Cas9 and functional assessment of targeted gene deletion on virulence can help improve our understanding of mechanisms of pathogenicity and represent the foundational framework that is necessary for development of these approaches. We propose to functionally delete 10 gene targets in Coccidioides posadasii strain Silveira and assay these deletions for loss of virulence. We will further investigate pathogenicity in C. immitis strain RS for those genes that show a loss of virulence. Currently, few specific mechanisms of virulence have been investigated in Coccidioides. Analysis of these data will provide new insights into functions of Coccidioides genes, which will increase our understanding of fungal pathogenesis mechanisms. Finally, we will work to develop tools to engineer fluorescently labeled live strains for Coccidioides using mCherry expression that will increase tools for interrogating host:pathogen interactions in an in vitro cell culture assay. In total, this proposal represents needed improvements in functional assessment of virulence in an important, yet understudied, fungal pathogen.

项目摘要/摘要 对大多数新出现的真菌病原体的毒力机制知之甚少。Coccidioids immitis和球藻C. 由于疾病迅速增加(球孢子菌病，又名谷热)，尤其令人担忧 美国的发病率，在过去20年中报告的疾病增加了10倍以上。《中心》 疾病控制和预防中心(CDC)的报告称，治疗山谷热的平均成本为每个患者5万美元， 包括长时间的住院和治疗方案。CRISPR/CAS9的使用及功能评价 针对毒力的靶向基因缺失有助于提高我们对致病机制和致病机制的理解 代表开发这些方法所必需的基本框架。我们建议 功能缺失Poscidioidosadasii Silveira株中的10个基因靶点并检测这些缺失的缺失情况 致命性。我们将进一步研究免疫球虫RS菌株的致病性，以寻找那些显示缺失的基因 致命性。目前，对球孢子虫的致病机制研究较少。分析 这些数据将为了解球孢子虫基因的功能提供新的见解，这将增加我们对 真菌致病机制的研究。最后，我们将致力于开发工具来设计荧光标记的LIVE 使用mCherry表达的球孢子虫菌株将增加询问宿主：病原体的工具 体外细胞培养试验中的相互作用。总而言之，这项提议代表了在功能上需要改进的地方 评估一种重要但未被充分研究的真菌病原体的毒性。