New Cleaver method accelerates engineering and genetics study of SARS-CoV-2 and its variants

A recent study published in bioRxiv*Server prepress characterization of rapid mutagenesis and rescue of SARS-CoV-2 variants without cloning, using a novel Reverse genetics strategy.

*Important note: bioRxiv It publishes preliminary scientific reports that have not been peer-reviewed and therefore should not be considered conclusive, directing clinical practice/health-related behaviour, or treated as hard information.

background

Well-established reverse genetics methods for coronaviruses were adapted to SARS-CoV-2 after it emerged. A DNA-based method, Infectious Sub-Genuine Amplicons (ISA), allows recombination of overlapping DNA fragments transcribed into a full-length genome, which was recently adapted for SARS-CoV-2.

Study and results

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

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

The team observed their first CPE five to eight days after transfection (DPT). They observed no improvements in virus recovery with co-transfection of a plasmid expressing nucleotides or messenger RNA (mRNA). Furthermore, as an improvement, the parts have been reduced from eight to four.

One virus-producing cell per 11,000 cells was observed by four-part transfection, compared to one per 160,000 cells in eight portions of transfection.

In addition, the team tested the effect of transfection using four fragments on recombination accuracy by comparing replication efficiency between wild-type isolates and recombinant viruses. The size and shape of wild-type virus and rCOV2 generated from four fragments (rCOV2-4fr) were similar.

Although the replication kinetics of rCOV2-4fr revealed decreased titers within 24 h post infection, titers were comparable after prolonged infection. Transmission electron microscopy was used to compare the viability of the virion. rCOV2-4fr was indistinguishable from wild-type virus.

The team indicated that the reconstitution process was reproducible, with successful recovery of virus from HEK293, HEK293T, BHK-21 and CHO cell lines.

Next, the team improved the amplification process by adopting some rules, such as using a high-fidelity template insertion or polymerase, restricting amplification cycles to 25, and assembling eight parallel amplification reactions.

The researchers introduced a genetic marker locus (Sal1 recognition) to distinguish between recombinant viruses and accidental contamination with clinical isolates. This marker was present in all recombinant viruses.

Furthermore, the team presented the genome sequence of the SARS-CoV-2 variants with/retaining the sequence of the remaining ancestral strain (Wuhan) as background.

The target sequence (variable spike gene) was amplified using commercial or endogenous plasmids. Chimeric viral 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. The titers were similar between chimeric viruses and their spiny counterparts.

An amplification step can be exploited for without mutants again Synthesis or reproduction. The team designed an oligonucleotide pair that inserted N501Y or G614D. The fragments harboring these point mutations were co-transfected with the other fragments required for genome assembly.

Sequencing confirmed the successful insertion of the substituents. Furthermore, they designed oligonucleotide primers for an ORF3a deletion, which was confirmed by sequencing and point immunoblotting.

Besides, the team succeeded in inserting an exogenous site-specific sequence (a triple FLAG tag) near the carboxy-end of ORF8. Moreover, the researchers slightly modified the strategy to rescue rCOV2 from clinical isolates directly.

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

One hundred base pairs of viral 3′ and 5′ untranslated regions (UTRs) surround the linker segment on both sides. Designed for successful recombination into a circular DNA product. Eight genomic fragments of SARS-CoV-2 were amplified in one step from viral RNA. The amplicon was transferred together with the linker fragment. Viable viruses were rescued at seven DPT.

This allowed the rescue of various chimeric viruses without bacterial cloning. The genomes of clinical isolates of the Wuhan SARS-CoV-2 strain and its Omicron variants (BA.1 and BA.5) were amplified.

The fragment containing the spike gene was exchanged with corresponding fragments carrying a variant spike gene(s). Thus, chimeric viruses carrying heterologous spike genes were generated.

Finally, the team rescued a recombinant virus from a clinical isolate of Omicron BA.5 as previously described, but using primers that introduced an ORF3a deletion. Likewise, recombinant XBB.1.5 with ORF3a deletion was rescued. Thus, the authors can generate deletion mutants of emerging SARS-CoV-2 variants in a single step.

conclusions

In short, Clever’s approach was highly versatile with wide application for rapid mutagenesis or SARS-CoV-2 rescue without cloning. The authors demonstrated the flexibility of this technique by introducing point mutations, foreign sequences, or ORF3a deletions.

Furthermore, by circulating intracellularly with the linker fragment, they introduced ORF3a deletion and rescued functional recombinants in one step of viral RNA.

*Important note: bioRxiv It publishes preliminary scientific reports that have not been peer-reviewed and therefore should not be considered conclusive, directing clinical practice/health-related behaviour, or treated as hard information.