Reverse genetics systems for SARS-CoV-2

Abstract

The ongoing pandemic of coronavirus disease 2019 (COVID-19) has caused severe public health crises and heavy economic losses. Limited knowledge about this deadly virus impairs our capacity to set up a toolkit against it. Thus, more studies on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) biology are urgently needed. Reverse genetics systems, including viral infectious clones and replicons, are powerful platforms for viral research projects, spanning many aspects such as the rescues of wild-type or mutant viral particles, the investigation of viral replication mechanism, the characterization of viral protein functions, and the studies on viral pathogenesis and antiviral drug development. The operations on viral infectious clones are strictly limited in the Biosafety Level 3 (BSL3) facilities, which are insufficient, especially during the pandemic. In contrast, the operation on the noninfectious replicon can be performed in Biosafety Level 2 (BSL2) facilities, which are widely available. After the outbreak of COVID-19, many reverse genetics systems for SARS-CoV-2, including infectious clones and replicons are developed and given plenty of options for researchers to pick up according to the requirement of their research works. In this review, we summarize the available reverse genetics systems for SARS-CoV-2, by highlighting the features of these systems, and provide a quick guide for researchers, especially those without ample experience in operating viral reverse genetics systems.

Keywords: BAC; CPER; SARS-CoV-2; TAR; reverse genetics systems.

Figure 1

Reverse genetics systems based on RNA recombination. Targeted RNA recombination was employed to generate the recombinant mouse hepatitis virus (MHV) genomic RNA. The synthetic donor RNA containing the S gene of feline infectious peritonitis virus (FIPV) is transcribed from the pFM1 vector, in which the FIPV S gene is flanked by the sequence derived from MHV. Then the donor RNA was transfected into mouse L2 cells, which were infected with the thermolabile MHV N gene deletion mutant. A crossover event within the HE gene fragment of the donor RNA happened, leading to the generation of the recombinant MHV genomic RNA with the FIPV S gene.

Figure 2

Reverse genetics system based on bacterial artificial chromosome (BAC). The genomic complementary DNA (cDNA) of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) without the S gene was separated into six component fragments. Fragment 1 (F1) was fused with a cytomegalovirus (CMV) promotor at its 5′ terminus, and the F6 was fused with poly(A) (pA), HDVr and BGH terminator at its 3′ terminus. SacII restriction site was inserted downstream of the TRS of S gene. All fragments have unique restriction sites at both ends. With these unique restriction sites, all the fragments were assembled into a pBAC‐MCS plasmid containing the designed restriction sites.

Figure 3

Reverse genetics system based on the in vitro ligation. Seven contiguous complementary DNA (cDNA) fragments covering the entire severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) genome were flanked by unique type IIS restriction endonucleases sites, which was adopted by No See′m cloning technology. F1 was fused with T7 promoter at its 5′ terminus. The fragments were cloned into a high‐copy plasmid for unlimited production. Each fragment was cleaved out of the plasmid with designed type IIS restriction endonucleases, purified from the gel, and ligated to each other to assemble the full‐length cDNA of SARS‐CoV‐2, which was transcribed with T7 polymerase to generate the infectious full‐length viral genomic RNA.

Figure 4

Reverse genetics system using yeast‐based TAR cloning. The genomic complementary DNA (cDNA) of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) was separated into twelve contiguous fragments with overlapping ends. F1 was fused with T7 promoter (red rectangle) and the overlapping sequence (light green rectangle), and F12 was fused with the other overlapping sequence (dark green rectangle). One‐step delivery of all the fragments and TAR vectors with the overlapping sequences (light green rectangle and dark green rectangle) were performed on yeast cells, and all DNA fragments were assembled by homologous recombination to generate the YAC vector containing the viral full‐length cDNA. The EagI site at the 5′ end of F12 was cleaved to linearize the vector. The linearized vector was used as the template to synthesize the viral infectious full‐length RNA.

Figure 5

Reverse genetics system based on circular polymerase extension reaction strategy (CPER). Ten contiguous fragments with overlapping ends covered the viral full‐length complementary DNA (cDNA) and were generated by polymerase chain reaction (PCR) with specific primers. A linker fragment containing the overlapping sequence with 3′ end of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2), the HDV ribosome, BGH/SV40 poly (A) signal, cytomegalovirus (CMV) promoter and the overlapping sequence with 5′ end of SARS‐CoV‐2, was designed to facilitate the circulation and the transcription of viral RNA in mammalian cells. All the DNA fragments that functioned as primers and templates in the same reaction system were amplified by PCR to generate circular DNA, which can be used to rescue the virus after being transfected in the package cells.