• The effect of nuclear localization signal tagging of recombinases/ integrases in recombinase-mediated cassette exchange efficiency in eukaryotes
  • Samaneh Ghanbari,1 Masoumeh Azizi,2 Pezhman Fard-Esfahani,3 Mohammad Hossein Modarressi,4 Fatemeh 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: Chinese hamster ovary (CHO) cells host over 70% of recombinant glycoproteins (1). CHO cell line development is mainly based on random integration (RI) of a gene of interest (GOI). Due to the uncontrollable integration sites, RI-generated cell pools are extremely heterogenous, requiring extensive screening to find stable and high-producing clones. Targeted integration of the GOI into a predefined locus has been introduced as a promising strategy to mitigate some limitations of the RI approach. Two main strategies have been used to achieve this goal. The first type involves the use of site-specific recombinases such as Cre/loxP and Flp/FRT in a technique known as recombinase-mediated cassette exchange (RMCE). The second type employs programmable nucleases, which CRISPR/Cas9 technology regards to be a breakthrough due to its efficiency, ease of use, and low cost. The generation of a platform cell line harboring a landing pad with a recombinase recognition site is a prerequisite of the RMCE technique (2,3). CRISPR/Cas9-mediated targeted integration of the landing pad into a predetermined locus of CHO cells has been proposed as a potential method for rational CHO platform cell line generation (4,5). Once the platform cell line has been established, it can be used to introduce any gene of interest using the RMCE technique. However, the low efficiency of the recombination event has remained an impediment to this approach. In this study, we reviewed several approaches that have been introduced to improve RMCE efficiency and therefore streamline the process of cell line development.
  • Methods: This study was a review and information was extracted from Google Scholar, PubMed, Science direct, and ProQuest databases by entering the desired keyword.
  • Results: Enhancing the nuclear transport of RMCE components, such as recombinase and donor plasmids, has been described as a potential way for improving RMCE efficiency. Shin et al. studied the effect of several types of nucleus localization signals (NLSs) and DNA nuclear-targeting sequences (DTSs) on recombinase and donor plasmid, respectively. Nucleoplasmin (NP) N-terminal NLS tagging of Cre recombinase and NP C-terminal NLS tagging of Bxb1 integrase showed approximately 4 times higher RMCE efficiency than that of the control group. On the other hand, NF-κB 5′/3′DTS was the most effective DTS in both Cre and Bxb1 RMCE systems (6). In another study, Andreas et al. investigated the effect of NLS on the performance of PhiC31 integrase and showed the strong enhancement of PhiC31 activity by the addition of C-terminal NLS (7). Xue et al investigated the effect of NLS addition in fourteen serin integrases in Saccharomyces cerevisiae and demonstrated the presence of NLS had little effect on most integrases’ efficiency except for TP901 and the φC31 integrases with slightly increased activity (8). Duportet et al. reported no improvement in integration efficiency upon NLS fusion to Bxb1 recombinase, albeit N-terminal NLS negatively impacts efficiency due to steric hindrance with catalytic domain (9).
  • Conclusion: In conclusion, reports of how fusing an NLS to recombinases affects RMCE efficiency have been contradictory. C-terminal NLS tagging may be advantageous for high-molecular-weight serine integrases like φC31 integrase that are unable to cross nuclear pores through passive diffusion. Acknowledgment: This work was financially supported by the Pasteur Institute of Iran (grant no. BD-9579) and National Institute for Medical Research Development (NIMAD’s project no. 978694)
  • Keywords: recombinase-mediated cassette exchange efficiency, nuclear localization signal, CHO cell line