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: 10818806
• 负责人: MAUREEN C FERRAN
• 金额: $ 1.91万
• 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: 2023-07-01 至 2025-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10818806
• 关键词: Acceleration; Administrative Supplement; American; Animals; Area; Biological Models; Biological Sciences; Biotechnology; CRISPR-mediated transcriptional activation; Caenorhabditis elegans; Caribbean region; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Communities; Development; Discipline; Disease; Foundations; Funding; Gene Activation; Gene Targeting; Genes; Genetic; Genetic Screening; Genomic approach; Goals; Grant; Guide RNA; Health; Individual; Intestines; Investigation; Laboratories; Large-Scale Sequencing; Libraries; Mediating; Mentorship; Messenger RNA; Methods; Modernization; Molecular; Nematoda; Organism; Pathway Analysis; Promoter Regions; Protein Isoforms; Qualifying; RNA; RNA Interference; Recombinants; Reporter Genes; Research; Research Project Grants; Scientist; Students; System; Systems Biology; Technology; Training; Transcription Coactivator; Transgenic Organisms; Underrepresented Populations; Variant; Vesicular stomatitis Indiana virus; Viral; Viral Genome; Viral Vector; Virion; Virus; Work; biological systems; career development; experimental study; expression vector; feeding; functional genomics; gene delivery system; gene discovery; genetic analysis; genetic approach; grasp; interest; large datasets; member; next generation; overexpression; promoter; screening; tool; undergraduate student; vector; viral vector development

Summary Page: Administrative Supplement Request to Promote Diversity in Health-Related Research (R21GM148859) Request: We are seeking an administrative supplement to promote diversity in health-related research for grant number R21GM148859. This funding would support Ms. Sydney Purcell, a 1st year Biotechnology and Molecular Biosciences major at the Rochester Institute of Technology (RIT}. Qualifications: Sydney qualifies for this supplement as a student from a group that is underrepresented in health-related research. Sydney identifies as a member of Caribbean American community. Overview of Sydney’s goals: This is a collaborative R21 grant and Sydney is working in Dr. Ferran’s laboratory at RIT. This research group is responsible for the research proposed in the first aim of the grant; therefore Sydney’s efforts will focus on the experiments underlined in the grant abstract below. As Sydney gains expertise she will help the next generation of students working on this project in the lab, therefore her trainees will also contribute to this work. Grant Abstract: 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 (C.) 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.

摘要页：促进健康相关研究多样性的行政补充请求 (R21GM148859) 请求：我们正在寻求行政补充，以促进健康相关研究的多样性 拨款号 R21GM148859 这笔资金将支持 Sydney Purcell 女士，她是生物技术和生物技术专业的一年级学生。 罗彻斯特理工学院 (RIT} 分子生物科学专业。 资格： 悉尼作为来自在该地区代表性不足的群体的学生，有资格获得此补助金 悉尼被认为是加勒比美洲社区的成员。 悉尼目标概述：这是一项 R21 合作资助，悉尼正在 Ferran 博士的实验室工作 RIT 的实验室负责该资助的第一个目标中提出的研究； 因此，悉尼的努力将集中在下面的资助摘要中强调的实验上。 获得专业知识，她将帮助下一代学生在实验室中从事这个项目，因此 她的学员也将为这项工作做出贡献。 格兰特摘要：实现现代系统生物学的承诺并掌握潜在的复杂性 生物系统的发展需要建立在高通量功能开发的基础上 能够生成大型数据集的基因组方法揭示了大规模测序工作。 相关性，但缺乏最好通过遗传方法提供的因果相互作用（C.）。 elegans 一直是基因发现和通路分析的主力，并且是唯一已建立的系统 可以在活的多细胞生物体的背景下进行高通量遗传分析 （即基于 RNAi 的喂养）尽管该模型系统很强大，但没有高通量方法可以实现。 该项目将探索如何在秀丽隐杆线虫中实现靶向基因过度表达。 两种不同病毒的重组株可以用作载体，以实现大规模遗传 线虫中基因过表达的分析 具体目标 1 的目标是实现启动子- 使用 CRISPRa 进行特异性基因激活。这种 CRISPR 变体形式依赖于切割缺陷亚型。 Cas9 (dCas9) 与转录激活剂融合，驱动目标基因的过度表达 具体来说，我们建议生成原理证明证据： 重组水泡性口炎病毒 (rVSV) 可以将 sgRNA 传递到转基因线虫中，该线虫表达 肠道细胞中的 CRISPRa 机制诱导 sgRNA 指导的报告基因过度表达。 我们的最终目标是开发一个针对启动子区域的综合 sgRNA VSV 文库 允许在线虫中进行高通量功能基因组筛选 具体目标 2 的目标是 开发奥赛病毒 (OV) 作为载体，将功能性 mRNA 外源传递到秀丽隐杆线虫中。 OV 作为基因传递系统很简单，因为这种病毒很容易通过肠道进入动物 简而言之，我们已经验证了表达 OV 基因组整合片段的线虫和线虫。 将使用这些现有菌株作为“包装系”来表达感兴趣的秀丽隐杆线虫基因 这些研究代表了初步的研究。 朝着使用 OV 作为过表达载体迈出一步，并将加速大规模开发 这些基于病毒的表达工具将整合这种多细胞生物体的规模遗传分析。 很容易使用线虫社区广泛使用的现有方法，这有可能 改变科学研究的多个领域，并对理解许多领域产生影响 疾病。