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.