New ‘CLEVER’ method accelerates engineering and genetic studies of SARS-CoV-2 and its variants

Recent research posted on Bio Rxiv*Preprint server described rapid mutagenesis and rescue of non-cloning severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants using a novel virus. reverse genetics strategy.

study: Rapid, cloning-free mutagenesis of novel SARS-CoV-2 variants using a novel reverse genetics platform. Image credit: kentoh/Shutterstock.com

*Important Notices: Bio Rxiv Publishes preliminary scientific reports that have not been peer-reviewed and should therefore not be considered definitive, to guide clinical practice or health-related actions, or to be treated as established information. not.

Background

Reverse genetics techniques established for CoV were also applied to post-emergence SARS-CoV-2. A DNA-based method, infectious subgenomic amplicon (ISA), which allows transfected overlapping DNA fragments to recombine into a full-length genome, was recently adapted for SARS-CoV-2.

research and discovery

In this study, researchers developed and described an ISA-based strategy called ‘Cloning-Free and Exchangeable System for Viral Engineering and Rescue’ (CLEVER) using SARS-CoV-2.

Eight overlapping SARS-CoV-2 genomic fragments were amplified and transfected into HEK293T cells. Growth of the resulting recombinant virus (rCOV2) was assessed by observing the cytopathic effect (CPE).

The research team observed the first CPE 5-8 days post-transfection (DPT). They observed no improvement in virus recovery by co-transfection of nucleotide-expressing plasmids or messenger RNA (mRNA). Additionally, as an optimization, the fragments have been reduced from 8 to 4.

1 virus-producing cell was observed per 11,000 cells when transfected with 4 fragments, compared to 1 per 160,000 cells when transfected with 8 fragments.

Additionally, the team tested the effect of transfection with the four fragments on reconstitution fidelity by comparing replication capacity between wild-type isolates and recombinant viruses. Wild-type virus and He generated rCOV2 from four fragments (rCOV2-4fr) were similar in size and shape.

Replication kinetics of rCOV2-4fr revealed a decrease in titer within 24 hours after infection, but titers were comparable even after prolonged infection. We compared virion integrity using transmission electron microscopy. rCOV2-4fr was indistinguishable from wild-type virus.

The research team successfully recovered virus from HEK293, HEK293T, BHK-21, and CHO cell lines, demonstrating that the reconstitution process was reproducible.

The team then optimized the amplification process by adopting several rules, including using high template input or high-fidelity polymerases, limiting amplification cycles to 25, and pooling eight parallel amplification reactions. bottom.

Researchers introduced a genetic marker (Sal I recognition) site to distinguish recombinant viruses from incidental contamination with clinical isolates. This marker was present in all rearranged viruses.

Additionally, the team introduced the spike gene sequence of the SARS-CoV-2 variant while maintaining/maintaining the sequence of the ancestral (Wuhan) strain remaining as background.

Target sequences (variant spike genes) were amplified using commercially available or in-house plasmids. Chimeric virus particles were rescued and confirmed by sequencing.

Rescued chimeric viruses or clinical isolates of the Wuhan strain and Omicron BA.1 or BA.5 variants were subjected to serum samples from vaccinated individuals. The highest neutralization titer was against the Wuhan strain. Titers between chimeric viruses and their respective spike homologs were similar.

An amplification step can be used for mutagenesis without any problems. again synthetic or cloned. The research team designed pairs of oligonucleotides that introduce N501Y or G614D. Fragments containing these point mutations were co-transfected with other fragments required for genome assembly.

Sequencing confirmed the successful introduction of the substitution. In addition, they designed oligonucleotide primers that deleted ORF3a, which were confirmed by sequencing and immunoblotting.

Furthermore, the research team successfully introduced a site-specific foreign sequence (triple FLAG tag) near the carboxy terminus of ORF8. In addition, the researchers slightly adjusted the strategy to directly rescue rCOV2 from clinical isolates.

They cloned the eukaryotic expression elements required for DNA-dependent transcription of the viral genome, or commercially available custom plasmids encoding these elements. All elements were assembled into a single linker DNA.

100 base pairs of viral 3′ and 5′ untranslated regions (UTRs) flanked both sides of the linker fragment. It was designed for successful recombination into circular DNA products. Eight SARS-CoV-2 genomic fragments were amplified in one step from viral RNA. Amplicons were co-transfected with the linker fragment. Viable virus was rescued in her 7 DPTs.

This made it possible to rescue various chimeric viruses without cloning the bacteria. The genomes of clinical isolates of SARS-CoV-2 Wuhan strain and Omicron variants (BA.1 and BA.5) were amplified.

Fragments containing the spike gene were replaced with corresponding fragments carrying the mutant spike gene. As a result, chimeric viruses carrying heterologous spike genes were generated.

Finally, the team rescued recombinant virus from a clinical isolate of Omicron BA.5 using primers that introduced the ORF3a deletion, as previously described. Similarly, recombinant XBB.1.5 with ORF3a deletion was also rescued. Thus, the authors were able to generate a new He SARS-CoV-2 mutant deletion mutant in one step.

Conclusion

In summary, the CLEVER approach was highly versatile and broadly applicable for rapid mutagenesis and rescue of SARS-CoV-2 without cloning. The authors demonstrated the flexibility of this technique by introducing point mutations, foreign sequences, or ORF3a deletions.

Furthermore, intracellular circularization using a linker fragment introduced the ORF3a deletion and rescued functional recombinant virus from viral RNA in one step.

*Important Notices: Bio Rxiv Publishes preliminary scientific reports that have not been peer-reviewed and should not be considered definitive, to guide clinical practice/health-related actions, or to be treated as established information. not.