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：
10431481
负责人：
PING WANG
金额：
$ 22.05万
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 2024-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10431481
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 Lead Libraries Life Molecular Molecular Disease Morphology Mucormycosis Mutagenesis Mutation Nutritional Requirements Pathogenesis Patients Phenotype Point Mutation Proteins RNA Research Research Project Grants Resistance Resources Rhizopus Sequence Homology Specificity System Time Virulence base 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%。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然未知。作为构建高通量全基因组的第一步。 R. delemar 突变体文库，我们建议生成小规模靶向突变体文库利用在特定目标1中，我们将合成一系列单向导的多重CRISPR/Cas9技术。 RNA (sgRNA) 以 40 个随机选择的基因为目标，并以一种方式提供汇集的 sgRNA 和 Cas9 在特定目标 2 中，我们将使用 Sanger DNA 识别基因突变。我们还将评估功效、特异性、多重 CRISPR/Cas9 诱导的脱靶突变是这项探索性研究的成功。致力于构建全基因组 CRISPR/Cas9 突变文库，用于系统性研究检查 R. delemar 发病机制。