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

项目摘要 实现现代系统生物学的承诺，把握生物系统的潜在复杂性 需要建立在高通量功能基因组方法的发展基础上， 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 最好是通过遗传途径。秀丽隐杆线虫一直是基因发现的主力， 途径分析，是唯一建立的系统，其中高通量遗传分析可以 在活的多细胞生物体的背景下进行（即基于摄食的RNAi）。尽管这东西的力量 模型系统，没有高通量的方法来实现靶基因在C.优雅的人 开发这个项目将探索两种不同病毒的重组株如何被改造成载体 以实现对C.优雅具体目标1的目的是 以使用CRISPRa实现启动子特异性基因激活。CRISPR的这种变体形式依赖于 Cas9的切割缺陷型同种型（dCas9）与转录激活因子融合以驱动Cas9的过表达， 由单基因RNA（sgRNA）靶向的基因。具体来说，我们建议生成原理证明 重组水泡性口炎病毒（rVSV）可以将sgRNA递送到转基因C. elegans 在肠细胞中表达CRISPRa机制以诱导sgRNA指导的报告基因过表达 基因最终，我们的目标是开发针对启动子区域的综合性sgRNA VSV文库， 允许在C中进行高通量功能基因组筛选。优雅具体目标2的目标是发展 利用奥赛病毒（OV）作为载体将功能性mRNA外源性地导入C.优雅使用OV作为 基因传递系统是直接的，因为这种病毒容易通过肠腔进入动物，并且C. 表达OV基因组的整合片段的秀丽线虫已经得到验证。简单地说，我们将使用这些 现有菌株作为“包装线”表达C.能够被整合到线虫中的感兴趣基因 新产生的病毒体感染受体线虫。这些研究是朝着使用 OV作为过表达载体，将加速这一领域大规模遗传分析的发展。 多细胞生物这些基于病毒的表达工具可以很容易地与现有的方法集成 广泛应用于C. elegans社区，这可能会改变多个领域的科学 研究，并对许多疾病的理解有影响。

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.