Rapid production of SARS-CoV-2 molecular clones using CRISPR-based yeast recombineering

使用基于 CRISPR 的酵母重组技术快速生产 SARS-CoV-2 分子克隆

• 批准号: 10247166
• 负责人: Hiten D Madhani
• 金额: $ 63.69万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-25 至 2022-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10247166
• 关键词: 2019-nCoV; A549; Affinity Chromatography; Antiviral Agents; Attenuated; CRISPR/Cas technology; Cell Line; Cells; Cloning; Clustered Regularly Interspaced Short Palindromic Repeats; Communities; Complementary DNA; Complex; DNA; Development; E protein; Electroporation; Engineering; Epithelial Cells; Escherichia coli; Family member; Foundations; Generations; Genes; Genetic Recombination; Genetic Transcription; Genome; Genomic DNA; Goals; Human; In Vitro; Infection; Innate Immune Response; Integration Host Factors; Investigation; Laboratories; Length; Luciferases; Lung; Mass Spectrum Analysis; Measures; Methods; Middle East Respiratory Syndrome; Molecular Cloning; Molecular Genetics; Molecular Virology; Monitor; Mutate; Mutation; Natural Immunity; Nonhomologous DNA End Joining; Nonstructural Protein; Open Reading Frames; Pathogenesis; Peptide Hydrolases; Phase; Plasmids; Process; Production; Proteins; Protocols documentation; RNA; RNA Caps; RNA replication; Replication Origin; Replicon; Reporter; Reporter Genes; Reporting; Research; Resources; Ribosomes; Role; SARS coronavirus; Saccharomyces cerevisiae; Series; Severe Acute Respiratory Syndrome; Site; Structural Genes; Structural Protein; Structure; Switzerland; System; T7 RNA polymerase; Tail; Testing; Variant; Viral; Viral Proteins; Viral Vaccines; Virus; Virus Replication; Work; Yeasts; base; design; experience; experimental study; homologous recombination; improved; multiple myeloma M Protein; mutant; nanoluciferase; pandemic disease; promoter; protein expression; rapid testing; repaired; synergism; tool; vaccine candidate; vector; viral RNA; virology

Objective: We propose to construct and distribute a series of SARS-CoV-2-derived molecular clones using powerful genome assembly and rapid CRISPR-based manipulation methods available in the yeast Saccharomyces cerevisiae. We will develop and apply methods to mutate and tag each of the 18 viral proteins in the context of a full-length viral cDNA clone from which virus can be produced and studied. Our two-PI UCSF team marries decades of experience in virology and yeast molecular genetics. Rationale: Coronaviral replication is a complex process involving numerous viral and host factors. While there is a strong foundation for studies of SARS-CoV-2 from prior studies of SARS, MERS, and other family members, there is no substitute for direct investigations of the virus responsible for the current pandemic. To date, there has been only one report of the generational of a full-length replicating molecular clone of SARS-CoV-2. In this approach, Thiel and colleagues in Switzerland used a yeast transformation-associated recombination (TAR) vector to assemble an infectious clone of SARS-CoV-2 from overlapping DNA fragments. The Madhani laboratory has 20 years of experience with recombinational cloning in yeast. We believe that the TAR approach can be rapidly improved and extended to generate a series of clones useful for investigation of viral RNA replication in a BSL2 context and the full viral cycle in a BSL3 context. The Andino lab has nearly 30 years of experience in molecular virology investigations. We propose to exploit the synergy offered by this team to rapidly develop, deploy and utilize a toolbox for investigations of SARS-CoV-2. Plan: To accomplish this goal we will 1) Improve the efficiency and utility of cloning in yeast. 2) Use transformation-associated recombination and rapid CRISPR-based yeast recombineering to generate series of molecular clones in S. cerevisiae derived from SARS-CoV-2. 3). Test the role of viral proteins in RNA replication and production of infectious virus. 4) Identify the protein interactome of viral proteins during a near-native infection cycle. Importantly, all clones and viruses will be made freely available to the research community. Impact: These resources and methods are anticipated to accelerate the development of rationally-engineered attenuated viral vaccine candidates, enable the rapid testing of antiviral compounds candidates using reporter viruses and increase fundamental understanding of the SARS-CoV-2 virus.

目的：我们建议使用以下方法构建和分发一系列 SARS-CoV-2 衍生的分子克隆 酵母中可用的强大的基因组组装和基于 CRISPR 的快速操作方法 酿酒酵母。我们将开发并应用方法来突变和标记 18 种病毒蛋白中的每一种 在全长病毒 cDNA 克隆的背景下，可以从中生产和研究病毒。我们的两位 PI UCSF 团队结合了数十年的病毒学和酵母分子遗传学经验。 理由：冠状病毒复制是一个复杂的过程，涉及许多病毒和宿主因素。在那里 之前对 SARS、MERS 和其他家族成员的研究为 SARS-CoV-2 研究奠定了坚实的基础， 对造成当前大流行的病毒进行直接调查是无可替代的。迄今为止，有 目前只有一份关于 SARS-CoV-2 全长复制分子克隆产生的报告。在这个 Thiel 和瑞士的同事使用了酵母转化相关重组 (TAR) 载体从重叠的 DNA 片段组装 SARS-CoV-2 的传染性克隆。迈达尼 实验室在酵母重组克隆方面拥有20年的经验。我们认为 TAR 方法 可以快速改进和扩展以生成一系列可用于研究病毒 RNA 的克隆 BSL2 环境中的复制和 BSL3 环境中的完整病毒周期。 Andino 实验室拥有近 30 年的经验 分子病毒学研究经验。我们建议利用该团队提供的协同作用，迅速 开发、部署和利用用于 SARS-CoV-2 调查的工具箱。 计划：为了实现这一目标，我们将 1) 提高酵母克隆的效率和实用性。 2）使用 转化相关重组和基于 CRISPR 的快速酵母重组工程可产生一系列 酿酒酵母中源自 SARS-CoV-2 的分子克隆。 3）。测试病毒蛋白在 RNA 复制中的作用 和传染性病毒的产生。 4) 鉴定近天然过程中病毒蛋白的蛋白质相互作用组 感染周期。重要的是，所有克隆和病毒都将免费提供给研究界。 影响：这些资源和方法预计将加速合理设计的开发 减毒病毒疫苗候选物，能够使用报告器快速测试抗病毒化合物候选物 病毒并增加对 SARS-CoV-2 病毒的基本了解。