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

• 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
• 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

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.

德氏根霉是威胁生命的毛霉菌病的主要原因 负责 约70%的临床病例，总死亡率超过50%。尽管 对人类健康的毁灭性影响， 很多R。德累马病理生物学与分子疾病 机制仍然未知。作为构建高通量全基因组 突变体库。delemar我们建议 为了产生小规模靶向突变体文库， 多重CRISPR/Cas9技术。在具体目标1中，我们将合成一组单指南 靶向40个随机选择的基因并在一个单克隆抗体中递送合并的sgRNA和Cas9的RNA（sgRNA）。 载体构建到R. delemar。在具体目标2中，我们将使用桑格DNA鉴定基因突变 测序和高通量下一代测序。我们还将评估疗效，特异性， 和由多重CRISPR/Cas9诱导的脱靶突变。这项探索性研究的成功 这项工作将导致构建全基因组CRISPR/Cas9突变体文库，用于系统性 对R. delemar发病机制。