Preliminary Study On CRISPR/Cas9-mediated Site-specific Knockin Of PCV2 Genes Encoding Functional Proteins

Porcine circovirus (PCV) belongs to the branch of Circovirus and the family of Circoviridae. PCV has two genotypes:porcine circovirus type 1(PCV1) is generally recognized as non-pathogenic to pigs, porcine circovirus type 2 (PCV2) which poses a huge threat to the porcine industry, has therefore aroused notable attention around the world. To approach the potential mechanim, we introduced gene expression cassettes of four PCV2 major functional proteins, alone or in combination, into porcine Rosa26 locus through CRISPR/Cas9-mediated knockin, thereby obtaining stable knock-in cell lines. It has laid a good foundation for exploring the underlying mechanisms of low productivity of PCV2 in mammalian cells. Currently, the major progresses include:1. Construction of two CRISPR/Cas9 plasmids targeting porcine Rosa26 locusDouble-strand DNA fragments were obtained by annealing two synthesized single-strand oligonucleotides and cloned into CRISPR/Cas9 vector. Sequencing result indicated the successful construction of CRISPR/Cas9 targeting porcine Rosa26 locus.2. Construction of four targeting (donor) vectors for homologous recombinationLeft homologous arm (LHA) and right homologous arm (RHA) were amplified by PCR using genomic DNA from PK15 cells as the templateand ligated into mpNTKV-Loxp vector, thereby referred to as mpNTKV-Loxp-LHA-RHA. Subsequently, four gene expression cassettes encoding PCV2 (Rep, Cap, Rep'), alone or in combination separated by IRES (internal ribozyme entry site), were cloned into mpNTKV-Loxp-LHA-RHA to obtain final targeting vectors, which were also confirmed by restrictive enzymes digestion and sequencing. Genomic DNA was perapared for T7 endonuclease I assay (T7E1). The results showed that each gRNA was able to induce NHEJ at its target site.3. Single cell colonies were obtained by drug screening following the cotransfection of CRISPR/Cas9 plasmid with donor vector into PK15 cellsCRISPR/Cas9 plasmids targeting Rosa26 locus, together with one ofthe four different recombinant donor vectors were co-transfected into PK15 cells by using lipofectamine 2000 as described by the mannual. Following the cotransfection, Drugs including G418 and Ganciclovir were applied to screen potential positive cell colonies. So far, total 192 cell colonies have been identified, and identification of positive clone by PCR universal testing primers had 18.In summary, this current work has layed an important foundation for exploring the underlying mechanisms of low productivity of PCV2 in PK15 cell and might facilitate the production of subunit vaccine of PCV2.