Chinese hamster ovary (CHO)
cells are regarded as a prominent host for manufacturing therapeutic
proteins. Although conventional strategies for generating recombinant
proteins in CHO
cells depend on the random integration of a gene of interest (GOI),
these established techniques occasionally result in genetically
heterogeneous cell lines, which causes diminished expression of the
recombinant proteins in the long run. Production instability can be
reduced by SSI and creates stable cell lines with a consistent
expression of the GOI. In this experiment, we demonstrate the targeted
incorporation of a reporter cassette in two PhiC31 pseudo attP sites of CHO
cells exploiting the homology-directed repair (HDR) generated by the
CRISPR/Cas9 platform. Genes encoding GFP and puromycin resistance marker
were precisely inserted into these loci via CRISPR/Cas9. Stable cell
lines were suitably produced following antibiotic selection. Junction
PCR and fluorescence assay determined targeted integration and expression
homogeneity of the reporter cassette, respectively. Taken together, our
results indicate the possibility of these two PhiC31 pseudo attP
sites as the target sites for site-specific integration of a transgene
mediated by CRISPR/Cas9. Furthermore, higher knock-in efficiency and
expression homogeneity was observed in the pseudo attP site associated with chromosome 6 compared to the pseudo attP site from chromosome 3.