tetano
Editor, Senior Moderator
Nat Protoc
. 2021 Jan 29.
doi: 10.1038/s41596-021-00491-8. Online ahead of print.
Engineering SARS-CoV-2 using a reverse genetic system
Xuping Xie[SUP] #[/SUP][SUP] 1 [/SUP], Kumari G Lokugamage[SUP] #[/SUP][SUP] 2 [/SUP], Xianwen Zhang[SUP] #[/SUP][SUP] 1 [/SUP], Michelle N Vu[SUP] 2 [/SUP], Antonio E Muruato[SUP] 1 2 [/SUP], Vineet D Menachery[SUP] 3 4 5 [/SUP], Pei-Yong Shi[SUP] 6 7 8 9 10 [/SUP]
Affiliations
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 cDNA fragments into a genome-length cDNA, (iv) in vitro transcribe RNA from the genome-length cDNA, (iv) electroporate the genome-length RNA into cells to recover recombinant viruses and (vi) characterize the rescued viruses. This protocol will enable researchers from different research backgrounds to master the use of the reverse genetic system and, consequently, accelerate COVID-19 research.
. 2021 Jan 29.
doi: 10.1038/s41596-021-00491-8. Online ahead of print.
Engineering SARS-CoV-2 using a reverse genetic system
Xuping Xie[SUP] #[/SUP][SUP] 1 [/SUP], Kumari G Lokugamage[SUP] #[/SUP][SUP] 2 [/SUP], Xianwen Zhang[SUP] #[/SUP][SUP] 1 [/SUP], Michelle N Vu[SUP] 2 [/SUP], Antonio E Muruato[SUP] 1 2 [/SUP], Vineet D Menachery[SUP] 3 4 5 [/SUP], Pei-Yong Shi[SUP] 6 7 8 9 10 [/SUP]
Affiliations
- PMID: 33514944
- DOI: 10.1038/s41596-021-00491-8
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 cDNA fragments into a genome-length cDNA, (iv) in vitro transcribe RNA from the genome-length cDNA, (iv) electroporate the genome-length RNA into cells to recover recombinant viruses and (vi) characterize the rescued viruses. This protocol will enable researchers from different research backgrounds to master the use of the reverse genetic system and, consequently, accelerate COVID-19 research.