مقالات پذیرفته شده در ششمین کنگره بین المللی زیست پزشکی
selection strategies for efficient enrichment of CHO cells in CRISPR Cas9 RMCE hybrid system
selection strategies for efficient enrichment of CHO cells in CRISPR Cas9 RMCE hybrid system
Samaneh Ghanbari,1Masoumeh Azizi,2Pezhman Fard-Esfahani,3Mohammad Hossein Modarressi,4Fatemeh Davami,5,*
1. Department of Medical Biotechnology, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran 2. Department of Molecular Medicine, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran 3. Department of Biochemistry, Pasteur Institute of Iran, Tehran, Iran 4. Department of Medical Genetics, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran 5. Department of Medical Biotechnology, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran
Introduction: Recombinant Chinese hamster ovary (CHO) cell line development (CLD) is a critical step for producing complex therapeutic glycoproteins. It is mainly based on random integration of the transgene of interest into the host genome followed by extensive screening to find stable high-producing clones. With the availability of the CHO genome and the advent of CRISPR/Cas9 technology, site-specific integration has been proposed as a potential method to address some drawbacks of the RI method. An intriguing method for accelerating CLD is the combination of CRISPR/Cas9 technology with recombinase-mediated cassette exchange (RMCE) technology. In the first step of this approach, platform cell line harboring a landing pad into a predefined locus of the host genome are generated utilizing the CRISPR/cas9 system. Then, employing the RMCE technology, any gene of interest can be inserted into the fully characterized platform cell line (1–3). In addition to the effectiveness of integration in both stages, selection strategies—which should effectively select cells that contain landing pads and enrich cells undergoing RMCE—are a key factor in achieving on-target clones quickly. Here we reviewed the selection methods applied to this end.
Methods: This study was a review and information were extracted from Google Scholar, PubMed, Science direct, and ProQuest databases by entering the desired keyword.
Results: The primary selection strategy for the first stage (platform cell line development) relies on positive selection utilizing antibiotics including puromycin, hygromycin, blasticidin, and G418 that are suitable for mammalian cell selection. Cells can be chosen with the appropriate antibiotic by including an antibiotic resistance gene in the landing pad design (4). The second strategy might involve using fluorescent activated cell sorting and including the genes for reporter proteins like GFP or mCherry into the landing pad. This method offers a selection approach devoid of antibiotics, which is very valuable for the creation of commercial cell lines (5–7).
In the second stage, cells with successful landing pad exchange with the gene of interest (GOI) should be enriched. Negative selection with ganciclovir (GCV) is one strategy provided that the thymidine kinase (TK) gene is incorporated into the landing pad to convert ganciclovir prodrug to its active form. Using this approach, it is anticipated that cells undergoing RMCE will survive after GCV selection (4,7). However, it has been noted that GCV counterselection does not completely eliminate all TK-positive cells (8,9). Therefore, it is crucial to take into account a variety of selection procedures for the second step. This step may also benefit from the aforementioned inclusion of the reporter gene into the landing pad. Indeed, cells with successful RMCE would be negative for the reporter protein and can be enriched using FACS system (5–7). Another selection strategy that has been reported as an efficient method to increase the enrichment of desirable cells is the promoter trap or poly (A) trap approach. With the promoter trap technique, the desired promoter is positioned outside of the landing pad, so that if the RMCE is accurate, a promoterless antibiotic resistance gene in the donor vector is inserted under the promoter, and desired cells survive after antibiotic selection. (7). There has also been report of using a combination of promoter and poly (A) trap. In this instance, a promoterless GOI RMCE donor vector was employed, ensuring that the GOI would only express upon the proper RMCE. In conjunction with the FACS enrichment method, this strategy can be an effective method for rapidly enriching cells that express a GOI (5).
Conclusion: The development of CHO cell lines using the CRISPR/Cas9 and RMCE combination is a two-step process that calls for an effective selection approach to enrich desired cells. Even if the effectiveness of targeted integration is low in the CRISPR or RMCE steps, stringent selection can compensate for this and eliminate any unwanted cells. As a result, combining selection options such as FACS enrichment and the promoter/poly (A) trap method could significantly shorten the CLD timeline.
This work was financially supported by Pasteur Institute of Iran (grant no. BD-9579) and National Institute for Medical Research Development (NIMAD’s project no. 978694)