Details

Autor(en) / Beteiligte

• Pourtabatabaei, Sana
• Ghanbari, Samaneh
• Damavandi, Narges
• Bayat, Elham
• Raigani, Mozhgan
• Zeinali, Sirous
• Davami, Fatemeh

Titel

Targeted integration into pseudo attP sites of CHO cells using CRISPR/Cas9

Ist Teil von

• Journal of biotechnology, 2021-08, Vol.337, p.1-7

Ort / Verlag

Elsevier B.V

Erscheinungsjahr

2021

Link zum Volltext

Quelle

Alma/SFX Local Collection

Beschreibungen/Notizen

• [Display omitted] •Pseudo attP sites are transcriptionally active sites for site-specific integration purposes.•Two pseudo attP sites of CHO-K1 cells are successfully targeted via CRISPR/Cas9 and compared in terms of level and stability of expression.•The pseudo attP site of chromosome 6 has higher knock-in efficiency and more homogenous expression than the pseudo attP site of chromosome 3. 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.