Development of FRESH (Fast Rescue Employing Self-Helper virus) - a rapid, generalizable method to rescue infectious virus from noninfectious genomic material

FRESH（使用自助病毒快速救援）的开发 - 一种从非感染性基因组材料中拯救感染性病毒的快速、通用方法

基本信息

批准号：
10089402
负责人：
Sanjay Vashee
金额：
$ 29.25万
依托单位：
J. CRAIG VENTER INSTITUTE, INC.
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-01-27 至 2023-12-31
项目状态：
已结题

项目摘要

While tools to manipulate viral nucleic acids have improved rapidly, methods to rescue infectious virus from genomic material that is not infectious have not. Methods for virus rescue are available for increasing numbers of virus species but must be individually developed and optimized for each different group of related viruses—a process that is time-consuming and costly. Therefore, there is a need to develop a generalizable synthetic genomics technology to facilitate rapid rescue of broad range of viruses at a reduced research cost to speed the development of genetic tools for novel, emerging, or understudied viruses. Here, we propose to develop FRESH (Fast Rescue Employing Self-Helper virus), a rescue strategy for viruses with noninfectious genomes and test its applicability for rescue of DNA and RNA viruses in prokaryotic and eukaryotic hosts. To boot infectivity of the genomic material with FRESH, a helper virus of the same virus species supplies trans-acting factors. In order to inhibit packaging of the helper virus, its genome is differentially targeted for CRISPR-Cas digestion. Sequence variation between the helper virus genome and the rescued genome, due to existing natural inter-strain variation or engineered mutations, confer immunity to the CRISPR guide RNAs. We have used FRESH to rescue African swine fever virus, which has a large DNA genome that is not infectious and no reported rescue methodology. To test the effectiveness of FRESH in a bacterial host, we propose to rescue P68, a virus of Staphylococcus, and inhibit the helper virus strain with CRISPR-Cas9 digestion. We will also develop FRESH with a Cas13 effector in mammalian cells to rescue human respiratory syncytial virus (RSV), as a model for negative-sense single- stranded RNA viruses. For each virus, we will determine the most effective guide RNAs, which will be used to inhibit the wild-type self-helper virus. Since we have already developed synthetic genomics tools for P68 and RSV, we will engineer the genomes to include a fluorescent reporter and to modify the sgRNA target regions, thus generating a CRISPR-immune reporter genome to be rescued. If these experiments are successful, additional work will be performed to define a straightforward FRESH rescue system that could be rapidly employed for previously undescribed viruses. Combined, FRESH and synthetic genomics can rapidly introduce sophisticated reverse genetics to virus species with noninfectious genomes allowing researchers to begin developing effective viral countermeasures in less time.

虽然操纵病毒核酸的工具迅速改善，但从没有传染性的基因组材料没有。病毒救援方法可用于增加数量病毒物种，但必须针对每个不同的相关病毒进行单独开发和优化 - A 耗时且昂贵的过程。因此，有必要开发可推广的合成基因组技术以降低的研究成本促进促进广泛病毒的快速营救，以加快开发用于新颖，新兴或理解病毒的遗传工具。在这里，我们建议开发新鲜（快速救援使用自助病毒），这是一种非感染基因组病毒的救援策略它适用于原核生物和真核宿主中DNA和RNA病毒的拯救。引导感染带有新鲜的基因组材料，是同一病毒物种的辅助病毒提供跨作用因子。为了抑制辅助病毒的包装，其基因组针对CRISPR-CAS消化的目标不同。顺序由于现有的自然应变变异，辅助病毒基因组和救出的基因组之间的变化或工程突变，赋予CRISPR指南RNA的免疫力。我们已经用新鲜来营救非洲人猪发烧病毒，具有大型DNA基因组，该基因组没有传染性，没有报道的救援方法。到在细菌宿主中测试新鲜的有效性，我们建议营救P68，即葡萄球菌病毒和用CRISPR-CAS9消化抑制辅助病毒菌株。我们还将使用CAS13效应器开发新鲜在哺乳动物细胞中拯救人呼吸道合胞病毒（RSV），作为负敏感的单个模型滞留的RNA病毒。对于每种病毒，我们将确定最有效的指南RNA，将用于抑制野生型自助病毒。由于我们已经为p68开发了合成基因组学工具 RSV，我们将设计基因组以包括荧光报告基因并修改SGRNA目标区域，从而产生CRISPR-Mumune报告基因组将被救出。如果这些实验成功，将进行额外的工作来定义一个直接的新鲜救援系统，这可能是迅速的用于以前未描述的病毒。结合，新鲜和合成的基因组学可以迅速引入软化的反向遗传学对具有非感染基因组的病毒物种，使研究人员可以开始在更少的时间内开发有效的病毒对策。