Fig. 1

Generation of recombinant SARS-CoV-2 by using an optimized CPER methodology. A Schematic of the optimized CPER system that includes new or modified steps including 5' end phosphorylation, nick ligation, as well as direct transfection of permissive cells with the CPER product. Specifically, the nine overlapping cDNA fragments (F1–F9/10) covering the full-length SARS-CoV-2 genome were phosphorylated at the 5′ end using T4 polynucleotide kinase (PNK) before being subjected to CPER assembly using a modified linker fragment (as illustrated in B). The circularized CPER product was then sealed at the staggered nicks by DNA ligation using HiFi Taq DNA ligase, and the closed circular infectious cDNA clone was transfected into Vero E6-TMPRSS2 cells for virus rescue. ‘P’ indicates phosphorylation. B Map of the linker plasmid (pGL-CPERlinker) in which the hepatitis delta virus (HDV) ribozyme, bovine growth hormone polyadenylation signal (bGH polyA), RNA polymerase II (Pol II) transcriptional pause site, and human cytomegalovirus (CMV) enhancer and promoter were assembled together with the ampicillin resistance (AmpR) cassette and the origin of replication (Ori) derived from the pUC19 plasmid (NEB). C Comparison of the optimized CPER system with the original co-culture method as described by Amarilla et al. [25] by rescuing a GFP reporter virus. Slight modifications to the ‘classical’ method, including the linker plasmid employed and the CPER thermocycling condition, were made to accommodate reagent unavailability and the 10-fragment genome segmentation scheme used in the optimized CPER method. GFP-positive syncytia were visible as early as day 3 and day 5 after CPER product delivery into Vero E6-TMPRSS2 cells and the HEK293T/Vero E6-TMPRSS2 co-culture system using the optimized and the classical CPER method, respectively. Of note, in the experiment using the optimized CPER workflow, the GFP signal declined on day 6 due to massive cell death. Scale bar, 100 μm. Data shown are representative of three independent experiments