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Towards a genome-wide CRISPR/Cas9 mutant library in Rhizopus delemar

德莱马根霉 (Rhizopus delemar) 中的全基因组 CRISPR/Cas9 突变体文库

基本信息

批准号：
10573271
负责人：
PING WANG
金额：
$ 18.38万
依托单位：
LSU HEALTH SCIENCES CENTER
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-02-15 至 2025-01-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10573271
关键词：
Amphotericin B CRISPR/Cas technology Clinical Code Collection Consumption DNA Polymerase III DNA sequencing Dideoxy Chain Termination DNA Sequencing Fungal Drug Resistance Future Gene Mutation Generations Genes Genetic Genome Genotype Glycine-Specific tRNA Growth Guide RNA Health Human Immunologic Deficiency Syndromes Induced Mutation Libraries Life Ligation Molecular Molecular Disease Morphology Mucormycosis Mutagenesis Mutation Nutritional Requirements Pathogenesis Patients Phenotype Point Mutation Proteins RNA Random Allocation Research Research Project Grants Resistance Resources Rhizopus Sequence Homology Specificity System Time Virulence design efficacy evaluation expression vector gene gun genome-wide improved innovation mortality mutant next generation sequencing off-target mutation pathogenic fungus promoter success tool vector whole genome

项目摘要

Rhizopus delemar is the primary cause of life-threatening mucormycosis responsible for approximately 70% of clinical cases, with an overall mortality rate exceeding 50%. Despite its devastating impact on human health, much of R. delemar’s pathobiology and molecular disease mechanisms remains unknown. As an initial step towards constructing a high-throughput genome-wide mutant library of R. delemar, we propose to generate a small-scale targeted mutant library utilizing multiplex CRISPR/Cas9 technology. In Specific Aim 1, we will synthesize a collection of single-guide RNAs (sgRNAs) targeting 40 randomly selected genes and deliver pooled sgRNAs and Cas9 in a one- vector construct into R. delemar. In Specific Aim 2, we will identify gene mutations using Sanger DNA sequencing and high-throughput next-generation sequencing. We will also assess efficacy, specificity, and off-target mutations induced by multiplex CRISPR/Cas9. The success of this exploratory research effort will lead to the construction of whole-genome CRISPR/Cas9 mutant libraries for the systemic examination of R. delemar pathogenesis mechanisms.

德莱马根霉是危及生命的毛霉菌病的主要原因负责约占临床病例的70%，总死亡率超过50%。尽管其对人类健康造成毁灭性影响， R. delemar 的大部分病理学和分子疾病机制仍不清楚。作为构建高通量全基因组的第一步 R. delemar 突变体文库，我们建议利用生成小规模靶向突变体库多重 CRISPR/Cas9 技术。在具体目标 1 中，我们将综合一系列单一指南 RNA (sgRNA) 以 40 个随机选择的基因为目标，并以单次方式提供汇集的 sgRNA 和 Cas9 将载体构建到 R. delemar 中。在具体目标 2 中，我们将使用 Sanger DNA 识别基因突变测序和高通量下一代测序。我们还将评估功效、特异性、以及多重 CRISPR/Cas9 诱导的脱靶突变。这项探索性研究的成功努力将导致构建全基因组 CRISPR/Cas9 突变体文库，用于系统性研究检查 R. delemar 发病机制。

项目成果

期刊论文数量（12）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Methionine biosynthesis enzyme MoMet2 is required for rice blast fungus pathogenicity by promoting virulence gene expression via reducing 5mC modification.

DOI：
10.1371/journal.pgen.1010927
发表时间：
2023-09
期刊：
PLoS genetics
影响因子：
4.5
作者：
通讯作者：

Cryptococcal Hsf3 controls intramitochondrial ROS homeostasis by regulating the respiratory process.

隐球菌 Hsf3 通过调节呼吸过程来控制线粒体内 ROS 稳态

DOI：
10.1038/s41467-022-33168-1
发表时间：
2022-09-15
期刊：
Nature communications
影响因子：
16.6
作者：
通讯作者：

Interplay between acetylation and ubiquitination of imitation switch chromatin remodeler Isw1 confers multidrug resistance in Cryptococcus neoformans.

DOI：
10.7554/elife.85728
发表时间：
2024-01-22
期刊：
eLife
影响因子：
7.7
作者：
Meng Y;Ni Y;Li Z;Jiang T;Sun T;Li Y;Gao X;Li H;Suo C;Li C;Yang S;Lan T;Liao G;Liu T;Wang P;Ding C
通讯作者：
Ding C

Homeostasis of cell wall integrity pathway phosphorylation is required for the growth and pathogenicity of Magnaporthe oryzae.