Viral vector-mediated gene activation to facilitate large-scale genetic analysis in Caenorhabditis elegans.

病毒载体介导的基因激活，以促进秀丽隐杆线虫的大规模遗传分析。

• 批准号: 10572507
• 负责人: MAUREEN C FERRAN
• 金额: $ 20.52万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-02-01 至 2025-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10572507
• 关键词: Acceleration; Aging; Animals; Area; Biological; Biological Models; Biological Phenomena; COVID-19 pandemic; CRISPR interference; CRISPR screen; CRISPR/Cas technology; Caenorhabditis elegans; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Communities; Complex; Development; Discipline; Disease; Double-Stranded RNA; Escherichia coli; Foundations; Gene Activation; Gene Expression; Gene Targeting; Genes; Genetic; Genetic Screening; Genomic approach; Goals; Guide RNA; Health; Human; Infection; Innate Immune Response; Intestines; Investigation; Large-Scale Sequencing; Libraries; Light; Low Density Lipoprotein Receptor; Mediating; Messenger RNA; Methods; Modernization; Natural Immunity; Nematoda; Open Reading Frames; Organism; Pathogenicity; Pathway Analysis; Phenotype; Population; Process; Production; Promoter Regions; Protein Isoforms; RNA; RNA Interference; RNA delivery; Reagent; Recombinants; Regulation; Reporter Genes; Research; System; Systems Biology; Technology; Testing; Tissues; Transcription Coactivator; Transgenic Animals; Transgenic Organisms; Variant; Vesicular stomatitis Indiana virus; Viral; Viral Genome; Viral Vector; Virion; Virus; Virus Diseases; Work; biological systems; cell type; expression vector; feeding; flexibility; follow-up; functional genomics; gene delivery system; gene discovery; genetic analysis; genetic approach; grasp; high throughput screening; improved; innovation; insight; interest; knock-down; large datasets; model organism; mutant; overexpression; pathogen; promoter; receptor; recombinant viral vector; screening; success; tool; uptake; usability; vector; virus host interaction

Project Summary Fulfilling the promise of modern systems biology and grasping the underlying complexity of biological systems requires a foundation built upon the development of high-throughput functional genomic approaches capable of generating large datasets. Large scale sequencing efforts reveal correlations, but lacks causal interactions best provided via genetic approaches. Caenorhabditis elegans has been a workhorse for gene discovery and pathway analysis, and is the only established system where high-throughput genetic analysis can be conducted in the context of a living multi-cellular organism (i.e. feeding based RNAi). Despite the power of this model system, no high-throughput methods to achieve targeted gene overexpression in C. elegans have been developed. This project will explore how recombinant strains of two different viruses can be adapted as vectors to enable large-scale genetic analysis of gene overexpression in C. elegans. The objective of Specific Aim 1 is to achieve promoter-specific gene activation using CRISPRa. This variant form of CRISPR relies on a cleavage defective isoform of Cas9 (dCas9) fused with a transcriptional activator to drive overexpression of a gene targeted by the single gene RNA (sgRNA). Specifically, we propose to generate proof-of-principle evidence that recombinant vesicular stomatitis virus (rVSV) can deliver a sgRNA into transgenic C. elegans that express the CRISPRa machinery in intestinal cells to induce sgRNA-directed overexpression of a reporter gene. Ultimately our goal is to develop a comprehensive sgRNA VSV library directed to promoter regions to allow high-throughput functional genomic screening in C. elegans. The objective of Specific Aim 2 is to develop Orsay virus (OV) as a vector to deliver functional mRNA exogenously into C. elegans. The use of OV as a gene delivery system is straightforward as this virus readily enters the animal via the intestinal lumen, and C. elegans expressing integrated segments of the OV genome have been validated. Briefly, we will use these existing strains as “packaging lines” to express C. elegans genes of interest capable of being incorporated in newly generated virion to infect recipient nematodes. These studies represent an initial step towards the use of OV as an overexpression vector and would accelerate the development large-scale genetic analysis in this multicellular organism. These viral-based expression tools would integrate easily with existing approaches widely used by the C. elegans community, which could potentially transform multiple areas of scientific investigation, and has implications for understanding of many diseases.

项目摘要 实现现代系统生物学的承诺并掌握生物系统的潜在复杂性 需要建立在能够开发的高通量功能基因组方法的基础上 生成大型数据集。大规模测序工作揭示了相关性，但缺乏因果相互作用 最好通过遗传方法提供。秀丽隐杆线虫一直是基因发现和 途径分析，是唯一可以进行高通量遗传分析的系统 在活的多细胞生物（即基于喂养的RNAi）的背景下进行。尽管有力量 模型系统，没有用于实现秀丽隐杆线虫中靶向基因过表达的高通量方法 发达。该项目将探讨如何将两种不同病毒的重组菌株作为向量调整 为了实现秀丽隐杆线虫中基因过表达的大规模遗传分析。特定目标的目的1是 使用CRISPRA实现特定于启动子的基因激活。这种变体的CRISPR依赖于 与转录激活剂融合的Cas9（DCAS9）的切割缺陷的同工型，以驱动A的过表达 由单个基因RNA（SGRNA）靶向的基因。具体而言，我们建议生成原理证明 重组囊泡气孔病毒（RVSV）可以将SGRNA输送到转基因秀丽隐杆线虫中的证据 表达肠细胞中的CRISPRA机械，以诱导记者的SGRNA定向过表达 基因。最终，我们的目标是开发一个针对发起人区域的全面的SGRNA VSV图书馆 允许在秀丽隐杆线虫中进行高通量功能基因组筛查。特定目标2的目的是发展 Orsay病毒（OV）作为载体，将功能mRNA外源性传递到秀丽隐杆线虫中。将OV用作 基因递送系统很简单，因为该病毒很容易通过肠腔进入动物，C。 表达OV基因组综合段的秀丽隐杆线已得到验证。简而言之，我们将使用这些 现有菌株作为表达能够纳入的感兴趣的秀丽隐杆线虫基因的“包装线” 新生成的病毒粒子被感染的线虫。这些研究是朝着使用的第一步 OV作为过表达的载体，并将加速开发大规模的遗传分析 多细胞生物。这些基于病毒的表达工具将轻松地与现有方法集成 秀丽隐杆线虫社区广泛使用，这可能会改变科学的多个领域 调查，对理解许多疾病具有影响。