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

虽然操纵病毒核酸的工具已经迅速改进，但从病毒中拯救感染性病毒的方法仍然存在。 没有传染性的基因组物质则没有。病毒拯救方法可用于越来越多的 但必须针对每一组不同的相关病毒进行单独开发和优化， 这是一个耗时且昂贵的过程。因此，需要开发一种可推广的合成药物。 基因组学技术，以降低研究成本，促进快速拯救广泛的病毒， 为新型、新兴或未充分研究的病毒开发遗传工具。在这里，我们建议开发FRESH (Fast采用自助病毒的拯救策略），这是一种针对非感染性基因组病毒的拯救策略， 其适用于原核和真核宿主中DNA和RNA病毒的拯救。为了靴子病毒的传染性 基因组材料与FRESH，一种相同病毒种类的辅助病毒提供反式作用因子。为了 抑制辅助病毒的包装，其基因组被差异靶向CRISPR-Cas消化。序列 辅助病毒基因组和拯救基因组之间的变异，由于现有的自然株间变异 或工程化突变，赋予对CRISPR指导RNA的免疫力。我们用FRESH拯救非洲人 猪瘟病毒，它具有大的DNA基因组，不具有传染性，也没有报道的拯救方法。到 为了测试FRESH在细菌宿主中的有效性，我们建议拯救P68，一种葡萄球菌病毒， 用CRISPR-Cas9消化抑制辅助病毒株。我们还将开发具有Cas 13效应器的FRESH 在哺乳动物细胞中拯救人呼吸道合胞病毒（RSV），作为负义单 单链RNA病毒对于每种病毒，我们将确定最有效的指导RNA，这些RNA将用于 抑制野生型自身辅助病毒。由于我们已经开发了针对P68的合成基因组学工具， RSV，我们将设计基因组以包括荧光报告基因并修饰sgRNA靶区域， 从而产生待拯救的CRISPR免疫报告基因组。如果实验成功， 将进行额外的工作，以确定一个简单的新鲜救援系统，可以迅速 用于以前未描述的病毒。FRESH和合成基因组学相结合， 对具有非传染性基因组的病毒物种进行复杂的反向遗传学研究，使研究人员能够开始 在更短的时间内开发有效的病毒对策。

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.