The production and application of SARS-CoV-2 reverse genetic systems to facilitate vaccine development and biosafe drug discovery platform

The development of coronavirus reverse genetic systems has been slow and limited to few laboratories worldwide. We have established a rapid approach to generate a SARSCoV-2 reverse genetic system, by targeted recombination of synthetic SARS-CoV-2 cDNA fragments in yeast, using a yeast artificial chromosome (YAC) vector. Nanopore minION sequencing was used to quickly identify correctly assembled YAC clones, that can be transcribed in vitro to produce RNA corresponding to the SARS-CoV-2 genome, which can be used to recover recombinant SARS-CoV-2 (Figure 1A). The entire process takes 2-3 weeks from the receipt of synthetic DNA. We now propose to use this reverse genetic system to develop: 1) recombinant SARS-CoV-2 reporter viruses expressing one or more reporter genes, in place of genes that are non-essential in cell culture: SARS-CoV-2 reporter viruses can be used to rapidly characterise viral infectivity and will be used to develop high-throughput virus antibody neutralisation and escape assays, to underpin viral vaccine development. 2) a biosafe SARS-CoV-2 "replicon" which lacks the viral structural genes but can replicate intracellularly. Human cell lines stably expressing SARS-CoV-2 replicons are powerful tools for antiviral screening and in contrast to assays with SARS-CoV-2, can be undertaken at containment level 2. These constructs will allow academic and commercial laboratories to undertake highthroughput antiviral assays, removing a bottleneck in drug discovery efforts. Furthermore, combined transcriptomics and proteomics will be used to characterise the effects of the recombinant reporter viruses and replicons on the host cell, ensuring that the engineered viruses faithfully represent the natural virus.