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Cre/Lox-based RMCE for Site-specific Integration in CHO Cells

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Abstract

Traditional cell line development is based on random genomic integration of transgenes. Random integration leads to unpredictable expression and results in clonal heterogeneity requiring a tedious screening procedure. Therefore, a new strategy is needed to establish clones that exhibit stable transgene expression. Here, we performed CRISPR/Cas9-mediated site-specific integration (SSI) to incorporate a landing pad (LP; containing mCherry) at a genomic hotspot (Fer1L4) allowing stable and strong expression. Site-specific integration of LP on Fer1L4 was demonstrated by sequencing results representing the swapped sequences in mCherry-expressing cells. We then performed Cre/Lox-based recombinase-mediated cassette exchange (RMCE) to exchange LP with a targeting vector (TV; containing GFP) in clones established by CRISPR/Cas9-mediated SSI. The success of Cre/Lox-based RMCE was evidenced by sequencing results representing the swapped sequences in GFP-expressing cells. Furthermore, the swapped clones expressing GFP was enriched up to 80%, indicating that the efficiency of Cre/Lox-based RMCE would be sufficient to swap pre-existing cassettes with gene-of-interest (GOI). Taken together, our study provides a new platform for Cre/Lox-based RMCE to iteratively establish stable clones from existing ones previously established by SSI at a genomic hotspot.

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References

  1. Kesik-Brodacka, M. (2018) Progress in biopharmaceutical development. Biotechnol. Appl. Biochem. 65: 306–322.

    Article  CAS  Google Scholar 

  2. Lai, T., Y. Yang, and S. K. Ng (2013) Advances in mammalian cell line development technologies for recombinant protein production. Pharmaceuticals (Basel). 6: 579–603.

    Article  CAS  Google Scholar 

  3. Carvalho, R. J. (2019) Comparison of cationic flocculants for the clarification of CHO-derived monoclonal antibodies. Biotechnol. Bioprocess Eng. 24: 754–760.

    Article  CAS  Google Scholar 

  4. Li, F., N. Vijayasankaran, A. Y. Shen, R. Kiss, and A. Amanullah (2010) Cell culture processes for monoclonal antibody production. MAbs. 2: 466–479.

    Article  Google Scholar 

  5. Coates, C. J., J. M. Kaminski, J. B. Summers, D. J. Segal, A. D. Miller, and A. F. Kolb (2005) Site-directed genome modification: derivatives of DNA-modifying enzymes as targeting tools. Trends Biotechnol. 23: 407–419.

    Article  CAS  Google Scholar 

  6. Hamaker, N. K. and K. H. Lee (2018) Site-specific integration ushers in a new era of precise CHO cell line engineering. Curr. Opin. Chem. Eng. 22: 152–160.

    Article  Google Scholar 

  7. Turan, S., C. Zehe, J. Kuehle, J. Qiao, and J. Bode (2013) Recombinase-mediated cassette exchange (RMCE) — a rapidly-expanding toolbox for targeted genomic modifications. Gene. 515: 1–27.

    Article  CAS  Google Scholar 

  8. Shin, S. W. and J. S. Lee (2020) CHO cell line development and engineering via site-specific integration: Challenges and opportunities. Biotechnol. Bioprocess Eng. 25: 633–645.

    Article  CAS  Google Scholar 

  9. Gidoni, D., V. Srivastava, and N. Carmi (2008) Site-specific excisional recombination strategies for elimination of undesirable transgenes from crop plants. In Vitro Cell. Dev. Biol. Plant. 44: 457–467.

    Article  CAS  Google Scholar 

  10. Schnütgen, F., A. F. Stewart, H. von Melchner, and K. Anastassiadis (2006) Engineering embryonic stem cells with recombinase systems. Methods Enzymol. 420: 100–136.

    Article  Google Scholar 

  11. Aach, J., P. Mali, and G. M. Church (2014) CasFinder: Flexible algorithm for identifying specific Cas9 targets in genomes. bioRxiv. 005074.

  12. Lee, J. S., T. B. Kallehauge, L. E. Pedersen, and H. F. Kildegaard (2015) Site-specific integration in CHO cells mediated by CRISPR/Cas9 and homology-directed DNA repair pathway. Sci. Rep. 5: 8572.

    Article  CAS  Google Scholar 

  13. Saraf-Levy, T., S. W. Santoro, H. Volpin, T. Kushnirsky, Y. Eyal, P. G. Schultz, D. Gidoni, and N. Carmi (2006) Site-specific recombination of asymmetric lox sites mediated by a heterotetrameric Cre recombinase complex. Bioorg. Med. Chem. 14: 3081–3089.

    Article  CAS  Google Scholar 

  14. Du, Z. W., B. Y. Hu, M. Ayala, B. Sauer, and S. C. Zhang (2009) Cre recombination-mediated cassette exchange for building versatile transgenic human embryonic stem cells lines. Stem Cells. 27: 1032–1041.

    Article  CAS  Google Scholar 

  15. Bouabe, H., R. Fässler, and J. Heesemann (2008) Improvement of reporter activity by IRES-mediated polycistronic reporter system. Nucleic Acids Res. 36: e28.

    Article  Google Scholar 

  16. Wang, B., T. Albanetti, G. Miro-Quesada, L. Flack, L. Li, J. Klover, K. Burson, K. Evans, W. Ivory, M. Bowen, R. Schoner, and P. Hawley-Nelson (2018) High-throughput screening of antibody-expressing CHO clones using an automated shaken deep-well system. Biotechnol. Prog. 34: 1460–1471.

    Article  CAS  Google Scholar 

  17. West, A. G. and P. Fraser (2005) Remote control of gene transcription. Hum. Mol. Genet. 14 Spec No 1: R101–R111.

    Article  CAS  Google Scholar 

  18. Turan, S., M. Galla, E. Ernst, J. Qiao, C. Voelkel, B. Schiedlmeier, C. Zehe, and J. Bode (2011) Recombinase-mediated cassette exchange (RMCE): traditional concepts and current challenges. J. Mol. Biol. 407: 193–221.

    Article  CAS  Google Scholar 

  19. Zhang, L., M. C. Inniss, S. Han, M. Moffat, H. Jones, B. Zhang, W. L. Cox, J. R. Rance, and R. J. Young (2015) Recombinase-mediated cassette exchange (RMCE) for monoclonal antibody expression in the commercially relevant CHOK1SV cell line. Biotechnol. Prog. 31: 1645–1656.

    Article  CAS  Google Scholar 

  20. Crawford, Y., M. Zhou, Z. Hu, J. Joly, B. Snedecor, A. Shen, and A. Gao (2013) Fast identification of reliable hosts for targeted cell line development from a limited-genome screening using combined φC31 integrase and CRE-Lox technologies. Biotechnol. Prog. 29: 1307–1315.

    Article  CAS  Google Scholar 

  21. Gaidukov, L., L. Wroblewska, B. Teague, T. Nelson, X. Zhang, Y. Liu, K. Jagtap, S. Mamo, W. A. Tseng, A. Lowe, J. Das, K. Bandara, S. Baijuraj, N. M. Summers, T. K. Lu, L. Zhang, and R. Weiss (2018) A multi-landing pad DNA integration platform for mammalian cell engineering. Nucleic Acids Res. 46: 4072–4086.

    Article  CAS  Google Scholar 

  22. Donnelly, M. L. L., L. E. Hughes, G. Luke, H. Mendoza, E. Ten Dam, D. Gani, and M. D. Ryan (2001) The ‘cleavage’ activities of foot-and-mouth disease virus 2A site-directed mutants and naturally occurring ‘2A-like’ sequences. J. Gen. Virol. 82: 1027–1041.

    Article  CAS  Google Scholar 

  23. Donnelly, M. L. L., G. Luke, A. Mehrotra, X. Li, L. E. Hughes, D. Gani, and M. D. Ryan (2001) Analysis of the aphthovirus 2A/2B polyprotein ‘cleavage’ mechanism indicates not a proteolytic reaction, but a novel translational effect: a putative ribosomal ‘skip’. J. Gen. Virol. 82: 1013–1025.

    Article  CAS  Google Scholar 

  24. Geier, M., P. Fauland, T. Vogl, and A. Glieder (2015) Compact multi-enzyme pathways in P. pastoris. Chem. Commun (Camb). 51: 1643–1646.

    Article  CAS  Google Scholar 

  25. Xia, T., S. Chen, Z. Jiang, Y. Shao, X. Jiang, P. Li, B. Xiao, and J. Guo (2015) Long noncoding RNA i suppresses cancer cell growth by acting as a competing endogenous RNA and regulating PTEN expression. Sci. Rep. 5: 13445.

    Article  CAS  Google Scholar 

  26. Inniss, M. C., K. Bandara, B. Jusiak, T. K. Lu, R. Weiss, L. Wroblewska, and L. Zhang (2017) A novel Bxb1 integrase RMCE system for high fidelity site-specific integration of mAb expression cassette in CHO cells. Biotechnol. Bioeng. 114: 1837–1846.

    Article  CAS  Google Scholar 

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Acknowledgments

This research was supported by priority Research Centers Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2020R1A6A1A0304195411). This research was also supported by Research Assistance Program (2019) in the Incheon National University.

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Authors and Affiliations

  1. Division of Life Sciences, College of Life Sciences and Bioengineering, Incheon National University, Incheon, 22012, Korea

    Jaewon Kim, Yun Haeng Lee, Myeong Uk Kuk, Su Young Hwang, Hyung Wook Kwon & Joon Tae Park

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  1. Jaewon Kim
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  2. Yun Haeng Lee
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  3. Myeong Uk Kuk
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  4. Su Young Hwang
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  5. Hyung Wook Kwon
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Contributions

KJW, HWK and JTP conceived of and designed the experiments. KJW, YHL, MUK and SYH performed the experiments. KJW and JTP analyzed the data. KJW, HWK and JTP wrote and edited the paper.

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Correspondence to Hyung Wook Kwon or Joon Tae Park.

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Kim, J., Lee, Y.H., Kuk, M.U. et al. Cre/Lox-based RMCE for Site-specific Integration in CHO Cells. Biotechnol Bioproc E 26, 795–803 (2021). https://doi.org/10.1007/s12257-020-0332-y

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  • DOI: https://doi.org/10.1007/s12257-020-0332-y

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