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.

摘要页面：行政补充请求，以促进与健康相关研究的多样性 （R21GM148859） 请求：我们正在寻求一种行政补充，以促进与健康相关研究的多样性 赠款号R21GM148859。这笔资金将支持悉尼Purcell女士，一年的生物技术和 罗切斯特理工学院（RIT}的分子生物科学专业。 资格：悉尼有资格获得该补充的资格，作为一名学生的学生的人数不足 与健康有关的研究。悉尼确定是加勒比美国社区的成员。 悉尼目标的概述：这是R21的合作，悉尼正在Ferran博士的工作 RIT实验室。该研究小组负责赠款的第一个目的提出的研究； 因此，悉尼的努力将重点放在下面的赠款摘要中强调的实验上。作为悉尼 获得专业知识，她将帮助下一代学生在实验室工作，因此 她的培训也将为这项工作做出贡献。 赠款摘要：实现现代系统生物学的承诺并掌握潜在的复杂性 生物系统需要建立在高通量功能的基础上的基础 能够生成大数据集的基因组方法。大规模的测序工作揭示了 相关性，但缺乏通过遗传方法最好提供的因果相互作用。 Caenorhabditis（C。） 秀丽隐杆线虫一直是基因发现和途径分析的主力军，并且是唯一建立的系统 在生活多细胞组织的背景下可以进行高通量遗传分析的地方 （即基于进食的RNAi）。尽管该模型系统具有力量，但仍未实现高通量方法 秀丽隐杆线虫中的靶向基因过表达已经开发出来。该项目将探讨如何 可以将两种不同病毒的重组菌株改编为载体，以实现大规模遗传 秀丽隐杆线虫中基因过表达的分析。特定目标的目的1是实现启动子 - 使用CRISPRA激活特定基因。 CRISPR的这种变体形式依赖于乳沟有缺陷的同工型 与转录激活剂融合的Cas9（DCAS9）的驱动基因的过表达 单基因RNA（SGRNA）。特别是，我们提议生成原则证据证明证据证明 重组囊泡口腔炎病毒（RVSV）可以将SGRNA输送到表达的转基因秀丽隐杆线虫中 肠细胞中的CRISPRA机械诱导记者基因的SGRNA指导的过表达。 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 开发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。使用 OV作为基因输送系统很简单，因为该病毒很容易通过肠道进入动物 表达OV基因组综合段的腔和秀丽隐杆线虫已得到验证。简而言之，我们 将这些现有菌株用作“包装线”，以表达能够成为的秀丽隐杆线虫基因 在新生成的病毒粒子中纳入感染受体线虫。这些研究代表了初始 迈向将OV用作过表达载体的迈向，并将加速发展。 在这个多细胞组织中的比例遗传分析。这些基于病毒的表达工具将集成 C.秀丽隐杆线虫社区广泛使用的现有方法很容易，这可能有可能 改变科学研究的多个领域，对理解许多 疾病。