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

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Principal Investigator: Hiten D Madhani
Organization: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
Fiscal Year: 2020
Award: $620,877
Funding agency: National Institute of Allergy and Infectious Diseases

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.

Terms: <2019 novel coronavirus><2019-nCoV><5' Capped RNA><5' mRNA Cap Structure><A549><Affinity Chromatography><Antiviral Agents><Antiviral Drugs><Antivirals><Attenuated><Baker's Yeast><Brewer's Yeast><CRISPR><CRISPR method><CRISPR methodology><CRISPR technique><CRISPR technology><CRISPR-CAS-9><CRISPR-based method><CRISPR-based technique><CRISPR-based technology><CRISPR-based tool><CRISPR/Cas method><CRISPR/Cas system><CRISPR/Cas technology><CRISPR/Cas9><CRISPR/Cas9 technology><Cas nuclease technology><Cell Body><Cell Line><CellLine><Cells><Cloning><Clustered Regularly Interspaced Short Palindromic Repeats><Clustered Regularly Interspaced Short Palindromic Repeats method><Clustered Regularly Interspaced Short Palindromic Repeats methodology><Clustered Regularly Interspaced Short Palindromic Repeats technique><Clustered Regularly Interspaced Short Palindromic Repeats technology><Communities><Complementary DNA><Complex><DNA><DNA Recombination><Deoxyribonucleic Acid><Development><E coli><E protein><E. coli><Electroporation><Engineering><Epithelial Cells><Escherichia coli><Esteroproteases><Family member><Foundations><Gene Transcription><Generations><Genes><Genetic Alteration><Genetic Change><Genetic Recombination><Genetic Transcription><Genetic defect><Genome><Genomic DNA><Goals><Host Factor><Host Factor Protein><Human><In Vitro><Infection><Innate Immune Response><Innate Immunity><Integration Host Factors><Investigation><Laboratories><Length><Luciferase Immunologic><Luciferases><Lung><Lung Respiratory System><M protein><MERS><MERS coronavirus disease><MERS-CoV disease><Mass Photometry/Spectrum Analysis><Mass Spectrometry><Mass Spectroscopy><Mass Spectrum><Mass Spectrum Analyses><Mass Spectrum Analysis><Measures><Methods><Middle East Respiratory Syndrome><Middle East Respiratory Syndrome CoV disease><Middle East Respiratory Syndrome coronavirus disease><Middle Eastern Respiratory Syndrome><Middle Eastern Respiratory Syndrome CoV disease><Middle Eastern Respiratory Syndrome coronavirus disease><Modern Man><Molecular Cloning><Molecular Genetics><Molecular Virology><Monitor><Mutate><Mutation><NHEJ><Native Immunity><Natural Immunity><Non-Homologous End Joining><Non-Polyadenylated RNA><Non-Specific Immunity><Non-homologous DNA End Joining><Non-structural Protein><Nonhomologous DNA End Joining><Nonhomologous End Joining><Nonspecific Immunity><Nonstructural Protein><ORFs><Open Reading Frames><Pathogenesis><Peptidases><Peptide Hydrolases><Phase><Plasmids><Process><Production><Protease Gene><Proteases><Protein Coding Region><Proteinases><Proteins><Proteolytic Enzymes><Protocol><Protocols documentation><RNA><RNA Caps><RNA Expression><RNA Gene Products><RNA replication><Recombination><Replication Origin><Replication Unit><Replicon><Reporter><Reporter Genes><Reporting><Research><Research Resources><Resources><Ribonucleic Acid><Ribosomes><Role><S cerevisiae><S. cerevisiae><SARS><SARS Virus><SARS corona virus><SARS coronavirus><SARS coronavirus disease><SARS-Associated Coronavirus><SARS-CoV><SARS-CoV disease><SARS-CoV-2><SARS-CoV2><SARS-Related Coronavirus><SARS-associated coronavirus 2><SARS-coronavirus-2><SARS-related coronavirus 2><Saccharomyces cerevisiae><Series><Severe Acute Respiratory Syndrome><Severe Acute Respiratory Syndrome CoV disease><Severe Acute Respiratory Syndrome Virus><Severe Acute Respiratory Syndrome corona virus><Severe Acute Respiratory Syndrome coronavirus><Severe Acute Respiratory Syndrome coronavirus disease><Severe acute respiratory syndrome coronavirus 2><Site><Strains Cell Lines><Structural Genes><Structural Protein><Structure><Switzerland><System><T7 RNA polymerase><Tail><Testing><Transcription><Variant><Variation><Viral><Viral Gene Products><Viral Gene Proteins><Viral Proteins><Viral Vaccines><Virus><Virus Replication><Work><Wuhan coronavirus><Yeasts><affinity purification><anti-viral agents><anti-viral drugs><anti-virals><base><cDNA><cultured cell line><design><designing><developmental><electroporative delivery><experience><experiment><experimental research><experimental study><gDNA><gene electrotransfer><genome mutation><homologous recombination><improved><multiple myeloma M Protein><mutant><nano-luciferase><nanoluciferase><ori Region><pandemic><pandemic disease><promoter><promotor><protein expression><pulmonary><repair><repaired><severe acute respiratory syndrome-CoV><social role><structural gene><synergism><tool><vaccine candidate><vector><viral RNA><viral multiplication><viral replication><virology><virus RNA><virus multiplication><virus protein>