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

> **NIH NIH R01** · UNIVERSITY OF CALIFORNIA, SAN FRANCISCO · 2021 · $636,859

## Abstract

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.

## Key facts

- **NIH application ID:** 10247166
- **Project number:** 3R01AI100272-10S1
- **Recipient organization:** UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
- **Principal Investigator:** Hiten D Madhani
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $636,859
- **Award type:** 3
- **Project period:** 2020-08-25 → 2022-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10247166

## Citation

> US National Institutes of Health, RePORTER application 10247166, Rapid production of SARS-CoV-2 molecular clones using CRISPR-based yeast recombineering (3R01AI100272-10S1). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10247166. Licensed CC0.

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