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

• 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
• 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
• 来源: https://reporter.nih.gov/project-details/10089402
• 关键词: Advanced Development; African Swine Fever Virus; Animals; Bacteria; Bacteriophages; Basic Science; Biological Models; Biology; CRISPR/Cas technology; Cell Culture Techniques; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Code; Complex; Consumption; DNA; DNA Viruses; Data; Development; Digestion; Ecosystem; Engineering; Genes; Genetic; Genome; Genome engineering; Genomics; Genus staphylococcus; Goals; Guide RNA; Health; Helper Viruses; Herpesviridae; Human; Human Microbiome; Immune; Immunity; Individual; Infection; Knowledge; Length; Mammalian Cell; Methodology; Methods; Modeling; Modification; Mutate; Mutation; Nucleic Acids; Plants; Plasmids; Play; Process; Production; Proteins; RNA; RNA Viruses; Reporter; Reporting; Research; Research Personnel; Respiratory syncytial virus; Role; Single Nucleotide Polymorphism; Source; Speed; Staphylococcus Phages; Staphylococcus aureus; System; Technology; Testing; Time; Trans-Activators; Translational Research; Variant; Viral; Viral Genes; Viral Genome; Virion; Virus; Work; cost; effectiveness testing; experimental study; genetically engineered virus; genome editing; genomic tools; improved; metagenomic sequencing; mutant; neglect; novel; pathogenic virus; rapid technique; reverse genetics; stable cell line; synthetic biology; synthetic genomics; tool; viral genomics; virology

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已经迅速改进，但拯救感染性病毒的方法