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

摘要页：促进健康相关研究多样性的行政补充请求 (R21GM148859) 要求：我们正在寻求行政补充，以促进与健康相关的研究的多样性 批准号为R21GM148859。这笔资金将支持悉尼·珀塞尔女士，生物技术专业一年级学生 罗切斯特理工学院分子生物科学专业。 资格：悉尼有资格获得这项补充，因为学生来自一个在 健康相关研究。悉尼认为自己是加勒比海美国人社区的一员。 悉尼的目标概述：这是一笔21兰特的合作赠款，西德妮正在费伦博士的 RIT的实验室。这个研究小组负责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拨款摘要中强调的实验上。作为西德妮 获得专业知识她将帮助实验室中从事这一项目的下一代学生，因此 她的实习生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抓住潜在的复杂性 生物系统的发展需要建立在高通量功能性开发的基础上 能够产生大型数据集的基因组方法。大规模测序工作揭示 相关性，但缺乏最好的通过遗传方法提供的因果互动。线虫(C.) Elgans一直是基因发现和路径分析的主力，也是唯一已建立的系统 高通量基因分析可以在活的多细胞生物体的背景下进行 (即基于摄食的RNAi)。尽管该模型系统功能强大，但没有实现高通量的方法 靶向基因在线虫中的过度表达已经被开发出来。本项目将探索如何 两种不同病毒的重组株可以作为载体，使大规模的基因 线虫中基因过表达的分析。具体目标1的目标是实现促进剂-- 使用CRISPRa激活特定基因。CRISPR的这种变异形式依赖于有切割缺陷的亚型 Cas9(DCas9)与转录激活子融合以驱动靶向基因的过度表达 单基因RNA(SgRNA)。具体地说，我们建议生成原则证明证据 重组水泡性口炎病毒(RVSV)可将sgRNA导入到表达sgRNA的转基因线虫中 肠道细胞中的CRISPRA机制诱导sgRNA引导的报告基因的过度表达。 最终，我们的目标是开发一个针对启动子区域的全面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目标是 发展Orsay病毒(OV)作为载体，将功能基因外源转移到线虫体内。对.的使用 OV作为一种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体内 Lumen，以及表达OV基因组整合片段的线虫已经得到验证。简而言之，我们 将使用这些现有的菌株作为“包装线”来表达感兴趣的线虫基因 加入到新产生的病毒粒子中，以感染受体线虫。这些研究代表了一项初步的 向使用OV作为过表达载体迈出一步，并将加速大规模- 在这种多细胞有机体中进行大规模遗传分析。这些基于病毒的表达工具将整合 很容易使用线虫社区广泛使用的现有方法，这可能会潜在地 改变了科学研究的多个领域，并对理解许多 疾病。

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.