Cryptococcus genomic resources

隐球菌基因组资源

基本信息

批准号：
10444326
负责人：
Hiten D Madhani
金额：
$ 76.37万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-06-01 至 2027-05-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10444326
关键词：
AIDS/HIV problem Accounting Animal Disease Models Animals Biochemical Biological C-terminal CRISPR screen CRISPR/Cas technology Cells Cessation of life Clinical Code Codon Nucleotides Collection Communities Coupled Cryptococcus Cryptococcus neoformans DNA DNA Sequence Data Data Analyses Databases Defect Deposition Diagnosis Disease Encapsulated Foundations Fungal Meningitis Gene Cluster Gene Deletion Gene Proteins Generations Genes Genetic Genome Genomics Growth Haploidy Human Image In Vitro Investigation Knock-out Laboratories Letters Libraries Maps Medical Methodology Methods Microscopy Modification Mus Mycoses Oligonucleotides Online Systems Open Reading Frames Pathway interactions Pharmaceutical Preparations Positioning Attribute Production Proteins Publications Quality Control Research Resources Saccharomyces cerevisiae Saccharomycetales Site The science of Mycology Update Virulence Work Yeast Model System base deletion library design design and construction fitness fungal genetics gene gun genome-wide homologous recombination knockout gene machine learning method member mutant next generation sequencing novel novel strategies pathogen pathogenic fungus screening therapy development tool web-accessible web-based informatics whole genome

项目摘要

Effective diagnosis and treatment of invasive fungal infections is a major unmet clinical challenge. Annotated genomic sequences of the major fungal pathogens create an opportunity to revolutionize medical mycology through the application of systematic approaches. To be maximally useful, genome sequences must be coupled with genome-wide biological resources. The availability of genome-wide knockout and tagged gene collections as well as a global genetic interaction map have been instrumental in catalyzing research progress in the model yeast Saccharomyces cerevisiae. The generation of analogous resources in pathogenic fungi creates experimental tools enabling comprehensive approaches to elucidating the basis of fungal virulence in the mammalian host and to develop drugs. Cryptococcus neoformans is an encapsulated budding yeast that causes fungal meningitis, resulting in ~200,000 fatalities each year. C. neoformans has haploid genetics, homologous recombination, and excellent animal models of disease. In the last period, we used biolistic transformation and long homology targeting constructs to complete a gene knockout collection, performed extensive quality control work, developed quantitative methods for screening the library in pools, and performed pooled screens both in mice and in vitro. We have made the strains available without restriction to the community via the Fungal Genetic Stock Center (FGSC). This resource has already been used in over 30 publications. To accelerate genome modification, we developed the first short-homology CRISPR-Cas9 methods for C. neoformans. We have also created an effective combined localization and purification tag. Preliminary work demonstrates that these methods can be deployed at scale to enable construction of a tagged strain collection. In addition, we have devised a novel method that enables pooled CRISPR-Cas9 screens in C. neoformans. Capitalizing on these technical advances, we will 1) complete the Cryptococcus whole-genome tag collection including production of an image resource, and 2) construct a first-generation genetic interaction map focused on virulence determinants. Accomplishment of the first aim will enable the cell biological and/or biochemical investigations of any protein. Completion of the second aim will deorphanize large fraction genes through membership in clusters of genes with known functions while defining new modules/pathways. This information will be integrated into the searchable and web accessible VEuPathDB database. Together these new genomic resources will accelerate the ability of the research community to understand and develop therapies against the most common cause of human fungal meningitis.

侵袭性真菌感染的有效诊断和治疗是尚未解决的重大临床挑战。带注释的主要真菌病原体的基因组序列为医学真菌学带来革命性的机会通过应用系统方法。为了发挥最大作用，基因组序列必须耦合拥有全基因组生物资源。全基因组敲除和标记基因集合的可用性以及全球遗传相互作用图有助于促进该模型的研究进展酿酒酵母。病原真菌中类似资源的产生创造了实验工具能够采用综合方法来阐明真菌毒力的基础哺乳动物宿主并开发药物。新型隐球菌是一种封装的芽殖酵母，可引起真菌性脑膜炎，每年导致约 20 万人死亡。 C. neoformans 具有单倍体遗传学，同源重组和优秀的疾病动物模型。在上一时期，我们使用了生物射弹变换和长同源靶向构建体以完成基因敲除收集，进行广泛的质量控制工作，开发了筛选库中文库的定量方法，并在小鼠和体外。我们通过真菌遗传向社区提供了不受限制的菌株库存中心（FGSC）。该资源已在 30 多种出版物中使用。加速基因组修改后，我们开发了第一个针对新型隐球菌的短同源 CRISPR-Cas9 方法。我们还有创建了一个有效的组合定位和纯化标签。初步工作表明，这些可以大规模部署方法以构建标记的菌株集合。此外，我们还有设计了一种新方法，可以在新型隐球菌中进行 CRISPR-Cas9 筛选。充分利用这些技术进步，我们将1）完成隐球菌全基因组标签收集，包括生产图像资源，2) 构建专注于毒力的第一代遗传相互作用图决定因素。第一个目标的实现将使细胞生物学和/或生化研究成为可能任何蛋白质。完成第二个目标将通过簇中的成员资格使大部分基因去孤儿化具有已知功能的基因，同时定义新的模块/途径。这些信息将被整合到可搜索且可通过网络访问的 VEuPathDB 数据库。这些新的基因组资源将共同加速研究界了解和开发针对最常见原因的治疗方法的能力人类真菌性脑膜炎。