Engineering SARS-CoV-2 using a reverse genetic system.

Nature Protocols
Xuping XiePei-Yong Shi

Abstract

Reverse genetic systems are a critical tool for studying viruses and identifying countermeasures. In response to the ongoing COVID-19 pandemic, we recently developed an infectious complementary DNA (cDNA) clone for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The reverse genetic system can be used to rapidly engineer viruses with desired mutations to study the virus in vitro and in vivo. Viruses can also be designed for live-attenuated vaccine development and engineered with reporter genes to facilitate serodiagnosis, vaccine evaluation and antiviral screening. Thus, the reverse genetic system of SARS-CoV-2 will be widely used for both basic and translational research. However, due to the large size of the coronavirus genome (~30,000 nucleotides long) and several toxic genomic elements, manipulation of the reverse genetic system of SARS-COV-2 is not a trivial task and requires sophisticated methods. Here, we describe the technical details of how to engineer recombinant SARS-CoV-2. Overall, the process includes six steps: (i) prepare seven plasmids containing SARS-CoV-2 cDNA fragment(s), (ii) prepare high-quality DNA fragments through restriction enzyme digestion of the seven plasmids, (iii) assemble the seven c...Continue Reading

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Oct 28, 2020·Nature·Jessica A PlantePei-Yong Shi

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Citations

Mar 26, 2021·Cell Reports·Izumi KimuraKei Sato
Jun 10, 2021·Nature Communications·Alberto A AmarillaAlexander A Khromykh
Jul 6, 2021·Bioinformatics and Biology Insights·Ritesh GorkhaliHitesh Kumar Bhattarai
Sep 19, 2021·Nature Reviews. Genetics·Kaiming TaoRobert W Shafer

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