Cryptococcus knockout and tag resource

人类危及生命的真菌感染的有效诊断和治疗是一个尚未满足的主要临床问题。 挑战。人类主要真菌病原体的注释良好的基因组序列已经在 几年来，创造了通过应用系统的 全基因组的方法。然而，为了最大限度地发挥作用，全基因组序列需要与 全基因组的生物资源。全基因组敲除和标记基因集合的可用性 在剖析模式酵母酿酒酵母的基本生物学方面发挥了重要作用。我们的 前提是在病原真菌中产生类似的集合将产生强大的实验 使综合方法能够阐明哺乳动物宿主中真菌毒力的基础的工具 并开发药物。这些工具通过应用现有的In来加速社区的研究 体内和体外测试可以从功能上描述所有基因和蛋白质在任何感兴趣的过程中的行为。 新生隐球菌是一种被包裹的芽生酵母，是引起真菌性脑膜炎的最常见原因。 据估计，每年约有100万例病例导致约60万人死亡。三分之一的艾滋病患者死亡 全世界都是隐球菌性脑膜脑炎的结果。这种重要的真菌病原体具有单倍体。 遗传和同源重组，使其成为优秀的模式病原体和模式生物。那里 有6967个预测的编码序列。在这笔赠款的最初阶段，我们开发了一种信息学 基础设施，设计和购买了约70,000个寡核苷酸，招聘/培训人员，并成功 构建并保存了4042株基因敲除菌株。我们估计 目前收集的基因涵盖了大约75%的非必需基因。我们已经用这个集合的一部分来 识别产生已知毒力因子所需的新基因。重要的是，我们已经制造了菌株 可通过真菌遗传库存中心向社区提供，不受限制。在这项修订的续期中 申请，我们将完成隐球菌基因缺失收集，进行另一轮质量- 包括Southern杂交在内的对照检查，完成我们对已知毒力属性的筛选，获得 在感染期间参考健康概况，然后利用我们的专业知识、试剂和基础设施来 构建一个菌株集合，其中每个编码蛋白质的基因都有双重表位标签，从而能够 对蛋白质功能的系统研究。这项工作的完成将在#年对外地产生巨大影响 为研究界提供任何人类病原体都无法获得的基因组资源。

The effective diagnosis and treatment of life-threatening fungal infections in humans is a major unmet clinical challenge. Well-annotated genomic sequences of the major human fungal pathogens have been available for a number of years, creating an opportunity to revolutionize medical mycology through the application of systematic genome-wide approaches. However, to be maximally useful, whole-genome sequences need to be coupled with the genome-wide biological resources. The availability of genome-wide knockout and tagged gene collections has been instrumental in dissecting the fundamental biology of the model yeast Saccharomyces cerevisiae. Our premise is that the generation of analogous collections in pathogenic fungi would create powerful experimental tools enabling comprehensive approaches to elucidating the basis of fungal virulence in the mammalian host and to develop drugs. Such tools accelerate research of the community through the application of existing in vivo and in vitro assays to functionally profile the behavior of all genes and proteins in any process of interest. Cryptococcus neoformans is an encapsulated budding yeast that is the most common cause of fungal meningitis. An estimated ~1,000,000 cases result in ~600,000 deaths each year. One-third of deaths in AIDS patients worldwide are the result of Cryptococcal meningoencephalitis. This important fungal pathogen has haploid genetics and homologous recombination, making it an excellent model pathogen and model organism. There are 6967 predicted coding sequences. In the initial period of this grant, we developed an informatics infrastructure, designed and purchased ~70,000 oligonucleotides, recruited/trained personnel and successfully constructed and deposited 4042 gene knockout strains using biolistic (“gene gun”) transformation. We estimate that the current collection covers ~75% of nonessential genes. We have used a portion of this collection to identify new genes required for the production of known virulence factors. Importantly, we have made strains available without restriction to the community via the Fungal Genetic Stock Center. In this amended renewal application, we will complete the Cryptococcus gene deletion collection, perform an additional round of quality- control checks including Southern hybridization, complete our screens for known virulence attributes, obtain reference profiles for fitness during infection, and then leverage our expertise, reagents and infrastructure to construct a strain collection in which each protein-coding gene is dual epitope-tagged, thereby enabling systematic studies of protein function. Completion of this work will have an enormous impact on the field by providing the research community with genomic resources not yet available for any human pathogen.