Engineering SARS-CoV-2 using a reverse genetic system

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.

Figure 1.. Overview of SARS-CoV-2 Reverse Genetic Systems.

The SARS-CoV-2 infectious clone model contains seven cDNA fragments to cover the complete viral genome, to disrupt toxic elements, and to aid in genetic manipulation. The SARS-CoV-2 plasmids are amplified in E. coli and sequentially ligated following digestion with type II restriction enzymes to remove the plasmid backbone. The full-length viral DNA is then in vitro transcribed using T7 polymerase to generate full-length genomic SARS-CoV-2 RNA and electroporated into cells with N-protein transcript expressed in trans. Following electroporation, cells are seeded into cell culture flasks and virus recovered 2–5 days post electroporation.

Figure 2.. Gel extraction of SARS-CoV-2 fragments, full-length cDNA, and full-length viral RNA.

(a) Agarose gel showing each SARS-CoV-2 DNA fragment (1μl of each) isolated post restriction enzyne digestion and utilized for in vitro ligation. B&C) Representative gels from successful (b) and unsuccessful (c) attempts to generate full-length SARS-CoV-2 cDNA. Representative agarose gels from successful (d) and unsuccessful (e) attempts to generate in vitro transcribed full-length SARS-CoV-2 viral RNA prior to electroporation and virus recovery.

Figure 3.. Representative gel images post restriction enzyme digestion.

The DNA ladders in bp are indicated. The corresponding fragments of SARS-CoV-2 restricted from the plasmids are outlined.

Figure 4.. Nine PCR amplicons prepared for Sanger sequencing.

The DNA ladders in bp are shown.

Figure 5.. Characterization of recombinant SARS-CoV-2.

(a) Bright-field images of the recombinant SARS-CoV-2 infected Vero E6 cells using EVOS M5000 Imaging System with 10x objective. Cytopathic effects appeared on day 2 after cells were inoculated with recovered SARS-CoV-2 P0 virus. (b) Sequence results of the recombinant SARS-CoV-2.