• Efficient site-specific integration of the GFP reporter gene employing linearized dsDNA CRISPR-Cas9 method, RNP approach, and a nano-delivery system in CHO cells
  • Shahin Amiri,1 Setare Adibzadeh,2 Arash Arashkia,3 Saeed Kaboli,4 Yousef Khazaei Monfared,5 Fatemeh Davami,6,*
    1. Department of Medical Biotechnology, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran
    2. Department of Medical Biotechnology, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran
    3. Virology Department, Pasteur Institute of Iran, Tehran, Iran
    4. Department of Medical Biotechnology, School of Medicine, Zanjan University of Medical Sciences, Zanjan, Iran.
    5. Department of Radiology, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA
    6. Department of Medical Biotechnology, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran


  • Introduction: The Chinese hamster ovary (CHO) cells are widely regarded as the ideal host system for the production of recombinant therapeutic proteins primarily because of their glycosylation pattern, which is similar to that of humans. The CHO cell line development (CLD) with high protein yields is among the industry's most challenging problems. For many years, the random integration approach was employed regularly for this purpose, however, because of the heterogenicity that results from no control over the integration sites, the productivity of the chosen clones gradually decreases over time. In contrast, targeted integration (TI) techniques produce the best outcomes when choosing clones with high transgene expression levels. In these methods, the transcriptionally active genomic regions are used for integration resulting in high and stable expression. Utilizing the CRISPR-Cas9 technology has revolutionized genome manipulation due to its wide range of applications including TI by inducing double-strand break (DSB). The mammalian cells repair the DSB through different pathways mostly the error-prone non-homologous end-joining (NHEJ) and precise homology-directed repair (HDR)-related genome editing processes. In the context of a repair, if any templates are provided, under certain conditions, the cell can integrate the donor template via the knock-in process. However, most mammalian cells favor NHEJ over the HDR pathway, CRISPR-mediated integration occurs at a rate of less than 10%, while significant attempts have been made to improve it. There are several approaches for increasing the efficiency of precise integration including donor design strategies. it has recently been demonstrated that utilizing the linearized double-strand donor could significantly increase HDR efficiency in mammalian cells. Additionally, several studies have shown that employing the Cas9/gRNA complex as ribonucleoproteins (RNPs) leads to a significant decrease in off-target effects in addition to an enhancement in TI efficiency. On the other hand, concerning using the other well-known delivery approaches such as electroporation and commercial reagents (lipofectamine, CRISPRMAX, etc.), implementing some nano polymers may be an appropriate choice for RNP delivery because of their very low cytotoxicity together with high delivery efficiency.
  • Methods: An in vitro RNP complex was made using the purified Cas9 protein and the sgRNA which was designed to target a specific hotspot on the CHO-K1 cell line genome. Characterized nanoparticles were combined with RNP complexes as a delivery strategy. To produce linearized donors by PCR using necessary primers, a plasmid donor including GFP and puromycin expression cassettes flanked by 1 kb left and right arms corresponding to the Chr6 hotspot site was used as the template. CHO-K1 cells were cultured under standard conditions seeded into the 24-well plate and then transfected with a nanoparticle/RNP mixture. Forty-eight hours post-transfection the cells were transferred into a 6-well plate and then incubated overnight. cells were then given the proper antibiotic concentration every two days for almost 14 days to establish a stable cell pool. To verify the presence of cells with the precise integration, 5′/3′ junction PCRs were performed to amplify the 5′ and 3′ junctions of the targeted locus and integrated transgene. limiting dilution processes were followed to achieve knock-in efficiency using individual clones. A two-sided Fisher's exact test was used to establish the statistical significance of the efficiency, which was reported as a percentage of 5′/3′ junction PCR-positive clones. Data with p<0.05 were considered significant.
  • Results: The stable cell pool was obtained and verified by 5′/3′ junction PCRs; The desired bands of approximately 1.4 kb and 1.5 kb were visualized on 1% agarose gel for 5′ and 3′ junction PCRs, respectively. Following clonal selection, the efficiency of transgene integration by the 5′/3′ junction PCR analysis of single-cell clones was evaluated through cell lysate procedures. the knock-in rate was high and approximately 51% of the recovered clones showed on-target integration (18 clones out of 35).
  • Conclusion: According to the results of the current experiment, the use of the in vitro linearized dsDNA CRISPR-Cas9 approach in conjunction with the RNP modality and the novel, extremely low toxic nano-based delivery vehicle significantly boosted HDR efficiency by up to 51%, making it comparable to the widely used commercial reagents.
  • Keywords: CRISPR/Cas9, RNP, targeted knock-in, nano-delivery, CHO