• Targeted Integration of a Reporter Cassette in pseudo-attP sites of CHO Cells using CRISPR/Cas9 system
  • Sana Pourtabatabaei,1 Narges Damavandi,2 Samaneh Ghanbari Mehrandooei,3 Sirous Zeinali,4 Fatemeh Davami,5,*
    1. Department of Biotechnology, College of Science, University of Tehran, Tehran, Iran
    2. Department of Biotechnology, College of Science, University of Tehran, Tehran, Iran
    3. Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran
    4. Kawsar Genomics and Biotech Center, Tehran, Iran
    5. Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran


  • Introduction: Chinese hamster ovary (CHO) cells are considered as a predominant host for the production of therapeutic proteins. Conventional methods for producing recombinant proteins in CHO cells are based on random integration of a gene of interest (GOI) followed by gene amplification. However, these conventional methods often result in cell lines with genetic heterogeneity and chromosomal instability which lead to reduced expression of the recombinant proteins over time. Site-specific integration of the desired genes overcomes these shortcomings and generates stable cell lines with the consistent expression of the GOI. Here we present targeted integration of a reporter cassette in two PhiC31 pseudo-attP sites of CHO cells using homology-directed repair (HDR) induced by the CRISPR/Cas9 system.
  • Methods: Cells Culture and Transfection: The CHO-K1 cells were maintained in DMEM/F12 medium supplemented with 10% fetal bovine serum and 10% CO2 at 37°C incubator. The Puromycin sensitivity was also determined. The day before transfection, approximately 1×105 cells/mL were cultured in 24-well tissue culture plates. Cells in each well were co-transfected with 250 ng of Donor plasmid and 250 ng of all-in-one plasmid mixed with 3.5 µL of DNAfectin in 50 µL of DMEM/F12. Stable cell lines generation: Two days after transfection, the cells were seeded in a 6-well plate and the cells were selected with 5 µg/mL of puromycin for approximately 2 weeks until a resistant pool was obtained. Stably transfected pools were then detected with 5′/3′ junction PCR. Cells were seeded into 96-well plates at a density of 1 cell/well in the absence of a selection antibiotic for the isolation of individual clones. On day 7, the single colonies were checked for GFP expression. Detecting targeted GFP expression: After identification by 5′/3′ junction PCR, positive clones were further analyzed for GFP expression by flow cytometry. For GFP expression, 1 ×106 cells of each clone were washed with PBS and re-suspended in 1 mL PBS containing 1% FBS for flow cytometry analysis.
  • Results: Plasmid construction and Design of guide RNAs: The candidate pseudo attP sites are intergenic sites and located on chromosomes 3 and 6 of Chinese hamsters. First, we designed three gRNAs against these pseudo attP sites of CHO cell genomes using CRISPOR online tool to be cloned into the all-in-one vector. Also, donor vectors encoding transgene expression cassettes flanked with 1 kb of genomic region around the candidate pseudo attP sites as 5’- and 3’- homology arms were designed. Development of an HDR mediated targeted integration platform for CHO cells using CRISPR/Cas9: To evaluate whether transgene cassettes can be integrated into the candidate pseudo attP site in a site-specific manner using the CRISPR/Cas9 system, the donor vector was co-transfected with all-in-one vector into CHO-K1 cells. Then, a stable cell pool was generated by puromycin selection of transfected cells for 2 weeks. After limited dilution, we obtained 144 single clones for pseudo attP site located in chromosome 6, among which 8 clones were GFP positive. Among these 8 GFP+ clones, 2 clones (clones 2 and 37) were 5’/3’ junction PCR positive and adequately expressed the target protein. These results indicate that targeted knock-in of the transgene cassettes was successfully achieved in these clones and knock-in efficiency was 25% for pseudo attP site of chromosome 6. Selection and identification of targeted stable cell clones: After transfection, we extracted genomic DNA and performed 5’/3’ junction PCR, which showed desired fragments in agarose gel analysis. In the stably transfected pools after 2 weeks of puromycin selection, the targeted integration group showed 99% of GFP expression. After verification of transfected pools expression, limiting dilution was used to obtain the targeted cell clones, which were further verified by 5’/3’ junction PCR for positive clones.
  • Conclusion: In this study, we identified an ideal Phic31 pseudo attP integration site and developed a reliable site-specific integration strategy mediated by CRISPR/Cas9 to rapidly develop stable recombinant CHO cell lines for biopharmaceutical industrial applications with high productivity and excellent stability. Using this approach, we successfully obtained stable cell lines to produce GFP and puromycin resistance gene with excellent cell stability and productivity. Our study demonstrates that the design and implementation of Cas9-sgRNA-based genome engineering are straightforward and fast.
  • Keywords: Chinese hamster ovary cell; Site-specific integration; CRISPR/Cas9 system; Homology directed repair;