tetano
Editor, Senior Moderator
mBio
. 2026 May 11:e0012226.
doi: 10.1128/mbio.00122-26. Online ahead of print.
A rapid yeast-based reverse genetics system reveals SARS-CoV-2 Omicron BA.2.86 variant spreads faster than Omicron JN.1 variant in primary human nasal and bronchial epithelial airway cultures
Jiayu Xu[SUP] #[/SUP][SUP] 1 [/SUP], Michelle Chamblee[SUP] #[/SUP][SUP] 1 [/SUP], Cheng Chih Hsu[SUP] #[/SUP][SUP] 1 [/SUP], Fei Jiang[SUP] 1 [/SUP], Phylip Chen[SUP] 2 [/SUP], Yuexiu Zhang[SUP] 1 [/SUP], Xueya Liang[SUP] 1 [/SUP], Amal O Amer[SUP] 3 [/SUP], Prosper N Boyaka[SUP] 1 [/SUP], Estelle Cormet-Boyaka[SUP] 1 [/SUP], Mark E Peeples[SUP] 2 4 5 [/SUP], Jianrong Li[SUP] 1 5 6 [/SUP]
Affiliations
Since the COVID-19 pandemic, several reverse genetics platforms for SARS-CoV-2 have been established. In general, a plasmid-based reverse genetics system is stable and easy to manipulate, distribute, and store. However, traditional methods for the assembly of a large viral genome in a plasmid rely on natural and artificially engineered restriction sites, which are inefficient, time-consuming, labor-intensive, and frequently not successful. Here, we developed a yeast-based homologous recombination system that allows the assembly of the SARS-CoV-2 genome as a cDNA in a bacterial artificial chromosome (BAC) plasmid in a single step. The entire protocol from cDNA construction to virus rescue is simple, rapid, accurate, highly efficient, and can be completed in 2 weeks. Using this system, we have quickly generated recombinant SARS-CoV-2 (rSARS-CoV-2) WA1, Omicron BA.2.86, and Omicron JN.1 viruses expressing mCherry, green fluorescent protein (GFP), and NanoLuc luciferase (Nluc) reporters. Insertion of these reporter genes does not significantly alter the replication of SARS-CoV-2 in cell culture. We also compared the replication kinetics of rSARS-CoV-2-WA1, BA.2.86, and JN.1 reporter viruses in ex vivo primary human nasal epithelial (HNE) and human bronchial epithelial (HBE) cultures. Omicron BA.2.86 replicated and spread more efficiently than JN.1, which spread much faster than SARS-CoV-2 WA1 in these cultures. In summary, we have developed a highly efficient yeast-based recombinant system for the construction of infectious cDNA clones of SARS-CoV-2, enabling rapid genetic manipulation of SARS-CoV-2. In addition, the reporter viruses generated in this study will be useful for monitoring SARS-CoV-2 infection in vitro and in vivo.IMPORTANCEReverse genetics systems are an essential tool for probing the biology of viruses, testing antivirals, and developing live-attenuated vaccines. However, it has been a challenge to generate a rapid reverse genetics system for coronaviruses. Here, we developed a rapid, highly efficient reverse genetics system for SARS-CoV-2 that uses yeast homologous recombination. In this procedure, overlapping DNA fragments encompassing the entire SARS-CoV-2 and BAC plasmid fragments containing a yeast replication origin were mixed and transformed into yeast cells to assemble infectious cDNA clones in a single step. This system has enabled us to rapidly generate nine SARS-CoV-2 viruses: WA1, Omicron BA.2.86, and JN.1 viruses each expressing one of three reporters for tracking virus infection in vitro and in vivo. This method is easy, convenient, and highly efficient, generating infectious cDNA clones within 2 weeks. This system could readily be adapted to construct infectious cDNA clones for other large RNA viruses.
Keywords: SARS-CoV-2; rapid reverse genetics system; virus spread.
. 2026 May 11:e0012226.
doi: 10.1128/mbio.00122-26. Online ahead of print.
A rapid yeast-based reverse genetics system reveals SARS-CoV-2 Omicron BA.2.86 variant spreads faster than Omicron JN.1 variant in primary human nasal and bronchial epithelial airway cultures
Jiayu Xu[SUP] #[/SUP][SUP] 1 [/SUP], Michelle Chamblee[SUP] #[/SUP][SUP] 1 [/SUP], Cheng Chih Hsu[SUP] #[/SUP][SUP] 1 [/SUP], Fei Jiang[SUP] 1 [/SUP], Phylip Chen[SUP] 2 [/SUP], Yuexiu Zhang[SUP] 1 [/SUP], Xueya Liang[SUP] 1 [/SUP], Amal O Amer[SUP] 3 [/SUP], Prosper N Boyaka[SUP] 1 [/SUP], Estelle Cormet-Boyaka[SUP] 1 [/SUP], Mark E Peeples[SUP] 2 4 5 [/SUP], Jianrong Li[SUP] 1 5 6 [/SUP]
Affiliations
- PMID: 42112831
- DOI: 10.1128/mbio.00122-26
Since the COVID-19 pandemic, several reverse genetics platforms for SARS-CoV-2 have been established. In general, a plasmid-based reverse genetics system is stable and easy to manipulate, distribute, and store. However, traditional methods for the assembly of a large viral genome in a plasmid rely on natural and artificially engineered restriction sites, which are inefficient, time-consuming, labor-intensive, and frequently not successful. Here, we developed a yeast-based homologous recombination system that allows the assembly of the SARS-CoV-2 genome as a cDNA in a bacterial artificial chromosome (BAC) plasmid in a single step. The entire protocol from cDNA construction to virus rescue is simple, rapid, accurate, highly efficient, and can be completed in 2 weeks. Using this system, we have quickly generated recombinant SARS-CoV-2 (rSARS-CoV-2) WA1, Omicron BA.2.86, and Omicron JN.1 viruses expressing mCherry, green fluorescent protein (GFP), and NanoLuc luciferase (Nluc) reporters. Insertion of these reporter genes does not significantly alter the replication of SARS-CoV-2 in cell culture. We also compared the replication kinetics of rSARS-CoV-2-WA1, BA.2.86, and JN.1 reporter viruses in ex vivo primary human nasal epithelial (HNE) and human bronchial epithelial (HBE) cultures. Omicron BA.2.86 replicated and spread more efficiently than JN.1, which spread much faster than SARS-CoV-2 WA1 in these cultures. In summary, we have developed a highly efficient yeast-based recombinant system for the construction of infectious cDNA clones of SARS-CoV-2, enabling rapid genetic manipulation of SARS-CoV-2. In addition, the reporter viruses generated in this study will be useful for monitoring SARS-CoV-2 infection in vitro and in vivo.IMPORTANCEReverse genetics systems are an essential tool for probing the biology of viruses, testing antivirals, and developing live-attenuated vaccines. However, it has been a challenge to generate a rapid reverse genetics system for coronaviruses. Here, we developed a rapid, highly efficient reverse genetics system for SARS-CoV-2 that uses yeast homologous recombination. In this procedure, overlapping DNA fragments encompassing the entire SARS-CoV-2 and BAC plasmid fragments containing a yeast replication origin were mixed and transformed into yeast cells to assemble infectious cDNA clones in a single step. This system has enabled us to rapidly generate nine SARS-CoV-2 viruses: WA1, Omicron BA.2.86, and JN.1 viruses each expressing one of three reporters for tracking virus infection in vitro and in vivo. This method is easy, convenient, and highly efficient, generating infectious cDNA clones within 2 weeks. This system could readily be adapted to construct infectious cDNA clones for other large RNA viruses.
Keywords: SARS-CoV-2; rapid reverse genetics system; virus spread.