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

项目摘要 兑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开发的基础上，能够 生成大型数据集。大规模测序工作揭示了相关性，但缺乏因果互动 最好是通过遗传方法提供。秀丽线虫一直是基因发现和研究的主力 途径分析，是唯一可以进行高通量基因分析的成熟系统 在活的多细胞生物体(即基于RNAi的摄食)的背景下进行。尽管它的力量如此强大 模型系统中，还没有高通量的方法来实现线虫中靶向基因的过度表达 发展起来的。该项目将探索如何将两种不同病毒的重组菌株改编为载体。 以实现对线虫基因过度表达的大规模遗传分析。具体目标1的目标是 利用CRISPRa实现启动子特异性基因激活。CRISPR的这种变体形式依赖于 切割缺陷的Cas9亚型(DCas9)与转录激活子融合以驱动a基因的过度表达 由单基因RNA(SgRNA)靶向的基因。具体地说，我们建议生成原则证明 重组水泡性口炎病毒(RVSV)可将sgRNA导入转基因线虫的证据 在肠道细胞中表达CRISPRA机制以诱导sgRNA指导的报告基因的过度表达 吉恩。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的目标是开发 奥赛病毒(OV)作为载体将功能基因外源导入线虫。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腔进入动物体内，而C. 表达卵子基因组整合片段的线虫已经得到验证。简单地说，我们将使用这些 现有的菌株可以作为“包装线”来表达感兴趣的线虫基因，能够被整合到 新产生的病毒粒子感染受体线虫。这些研究代表着朝着使用 OV作为过表达载体，将加速本研究大规模遗传分析的发展。 多细胞生物体。这些基于病毒的表达工具可以很容易地与现有方法集成 被线虫群落广泛使用，这可能会改变科学的多个领域 调查，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.