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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

基本信息

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
项目类别：
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

项目摘要

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：构建和分布一系列sars - cov -2衍生的分子克隆