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

Objective: We propose to construct and distribute a series of SARS-CoV-2-derived molecular clones usingpowerful genome assembly and rapid CRISPR-based manipulation methods available in the yeastSaccharomyces cerevisiae. We will develop and apply methods to mutate and tag each of the 18 viral proteinsin the context of a full-length viral cDNA clone from which virus can be produced and studied. Our two-PI UCSFteam marries decades of experience in virology and yeast molecular genetics.Rationale: Coronaviral replication is a complex process involving numerous viral and host factors. While thereis 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, therehas been only one report of the generational of a full-length replicating molecular clone of SARS-CoV-2. In thisapproach, 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 Madhanilaboratory has 20 years of experience with recombinational cloning in yeast. We believe that the TAR approachcan be rapidly improved and extended to generate a series of clones useful for investigation of viral RNAreplication in a BSL2 context and the full viral cycle in a BSL3 context. The Andino lab has nearly 30 years ofexperience in molecular virology investigations. We propose to exploit the synergy offered by this team to rapidlydevelop, 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) Usetransformation-associated recombination and rapid CRISPR-based yeast recombineering to generate series ofmolecular clones in S. cerevisiae derived from SARS-CoV-2. 3). Test the role of viral proteins in RNA replicationand production of infectious virus. 4) Identify the protein interactome of viral proteins during a near-nativeinfection 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-engineeredattenuated viral vaccine candidates, enable the rapid testing of antiviral compounds candidates using reporterviruses and increase fundamental understanding of the SARS-CoV-2 virus.