CRISPR/Cas mediated disruption of the Porcine Circovirus-1 gene present in PK-15 cells using sgRNA cocktail

Porcine circovirus 1(PCV-1) is an inherent contaminant in all cells of porcine origin, including cell lines. Porcine Kidney 15 (PK-15) cells are one such cell line which are widely being used for the propagation of different porcine viruses and development of cell culture vaccines for pigs. PCV1 is a single-stranded DNA virus that remain in circular form with a high copy number inside the host cell. The presence of PCV1virus in PK-15 cells may affect the yield of porcine viruses and other vaccine strains like Classical Swine Fever Virus (CSFV) propagated in these cells. Hence the present study was conducted to explore the promising CRISPR/Cas9 tool in cleaving PCV-1 DNA from PK-15 cells, followed by the evaluation of these cells for producing CSF vaccine virus with better yield. PK-15 cells were subjected to CRISPR/Cas9 mediated cleavage with a cocktail of guide RNAs (sgRNAs) to cleave PCV-1 DNA. A significant reduction of PCV-1 DNA in the transfected cells was observed in droplet digital PCR and real-time PCR; indicating successful targeting of PCV-1 DNA by the chosen sgRNAs. Further, the PCV-1 cleaved PK-15 cells were infected with CSFV and found to have a better yield of harvested virus. Based on the present study, it can be suggested that knocking out of PCV-1 DNA from PK-15 cells offers a promising platform for generating contaminant free cell lines and vaccine development with higher yield.