A recent study published on the bioRxiv preprint server describes a new, cloning-free reverse genetics strategy for mutagenesis and rescue of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants. Previous established reverse genetics methods for coronaviruses were adapted to SARS-CoV-2 after its emergence. The new strategy, called “cloning-free and exchangeable system for virus engineering and rescue” (CLEVER), uses infectious subgenomic amplicons (ISA) to recombine transfected overlapping DNA fragments into a full-length genome, which was recently adapted to SARS-CoV-2.
The CLEVER approach is highly versatile with a broad application for rapid mutagenesis or rescue of SARS-CoV-2 without cloning. The researchers introduce optimization methods to improve the process including reducing the number of fragments required and testing the impact of transfection using four fragments specially designed to compare replication competence between wild-type isolates and recombinant viruses. Replication kinetics of the four fragment recombinant virus revealed reduced titers within 24 hours post-infection, but titers were comparable after prolonged infection.
Using CLEVER, researchers introduced a genetic marker site to differentiate recombinant viruses from accidental contamination with clinical isolates and successfully introduced N501Y or G614D point mutations through oligonucleotide pairs. They also designed oligonucleotide primers to delete the open reading frame 3a (ORF3a) gene, which was successfully confirmed by sequencing and immunoblot. Besides, the team successfully introduced a site-specific foreign sequence (triple FLAG tag) near the carboxy end of ORF8.
Furthermore, the researchers slightly tweaked the strategy to rescue rCOV2 from clinical isolates directly by cloning eucaryotic expression elements required for DNA-dependent transcription of the viral genome or commercially obtained custom plasmids encoding these elements. The rescue was successful, which allows the rescue of chimeric viruses without bacterial cloning. The strategy was flexible and convenient in introducing point mutations, foreign sequences, or ORF3a deletion, thus generating deletion mutants of emerging SARS-CoV-2 variants in a single step.
Overall, the new method allows for rapid mutagenesis and rescue of SARS-CoV-2 variants without the need for cloning, making the process more efficient and faster. However, it is important to note that the study is a preprint and not peer-reviewed, which means that the findings should not be regarded as conclusive, guide clinical practice/health-related behavior or treated as established information.
In summary, the CLEVER method is a significant step forward in SARS-CoV-2 engineering and genetic study. The new strategy enables improved efficiency in the genetic manipulation of SARS-CoV-2 and its variants, which could ultimately lead to a better understanding of the virus and its variants, as well as the development of more effective treatments and vaccines.
