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：我们建议使用使用一系列SARS-COV-2衍生的分子克隆来构建和分发 强大的基因组组件和基于CRISPR的快速操纵方法可在酵母中使用 酿酒酵母。我们将开发和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，可以从中产生和研究病毒。我们的两个PI UCSF 团队在病毒学和酵母分子遗传学方面拥有数十年的经验。 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。那里 是对SARS，MERS和其他家庭成员的先前研究研究SARS-COV-2的坚实基础， 无法直接研究导致当前大流行的病毒。迄今为止，那里 仅是SARS-COV-2的全长复制分子克隆的世代的一份报道。在这个 瑞士的Thiel及其同事使用酵母转化相关的重组（TAR） 从重叠的DNA片段中组装出SARS-COV-2的传染性克隆。 Madhani 实验室在酵母中具有20年的重组克隆经验。我们相信焦油方法 可以快速改进并扩展以产生一系列用于研究病毒RNA的克隆 在BSL2上下文中的复制和BSL3上下文中的完整病毒周期。 Andino实验室有近30年 有分子病毒学研究的经验。我们建议利用该团队提供的协同作用来迅速 开发，部署和利用一个工具箱来调查SARS-COV-2。 计划：为了实现这一目标，我们将1）提高克隆在酵母中的效率和实用性。 2）使用 与转化相关的重组和快速基于CRISPR的酵母重新组合，以产生一系列 酿酒酵母中的分子克隆来自SARS-COV-2。 3）。测试病毒蛋白在RNA复制中的作用 和传染病的产生。 4）确定近本性期间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社区。 影响：预计这些资源和方法将加速合理设计的发展 减毒病毒疫苗候选物，可以使用记者快速测试抗病毒药物。 病毒并增加对SARS-COV-2病毒的基本了解。