Applied Microbiology and Biotechnology

, Volume 102, Issue 14, pp 6105–6117 | Cite as

Rapid development of stable transgene CHO cell lines by CRISPR/Cas9-mediated site-specific integration into C12orf35

  • Menglin Zhao
  • Jiaxian Wang
  • Manyu Luo
  • Han Luo
  • Meiqi Zhao
  • Lei Han
  • Mengxiao Zhang
  • Hui Yang
  • Yueqing Xie
  • Hua Jiang
  • Lei Feng
  • Huili Lu
  • Jianwei Zhu
Applied genetics and molecular biotechnology


Chinese hamster ovary (CHO) cells are the most widely used mammalian hosts for recombinant protein production. However, by conventional random integration strategy, development of a high-expressing and stable recombinant CHO cell line has always been a difficult task due to the heterogenic insertion and its caused requirement of multiple rounds of selection. Site-specific integration of transgenes into CHO hot spots is an ideal strategy to overcome these challenges since it can generate isogenic cell lines with consistent productivity and stability. In this study, we investigated three sites with potential high transcriptional activities: C12orf35, HPRT, and GRIK1, to determine the possible transcriptional hot spots in CHO cells, and further construct a reliable site-specific integration strategy to develop recombinant cell lines efficiently. Genes encoding representative proteins mCherry and anti-PD1 monoclonal antibody were targeted into these three loci respectively through CRISPR/Cas9 technology. Stable cell lines were generated successfully after a single round of selection. In comparison with a random integration control, all the targeted integration cell lines showed higher productivity, among which C12orf35 locus was the most advantageous in both productivity and cell line stability. Binding affinity and N-glycan analysis of the antibody revealed that all batches of product were of similar quality independent on integrated sites. Deep sequencing demonstrated that there was low level of off-target mutations caused by CRISPR/Cas9, but none of them contributed to the development process of transgene cell lines. Our results demonstrated the feasibility of C12orf35 as the target site for exogenous gene integration, and strongly suggested that C12orf35 targeted integration mediated by CRISPR/Cas9 is a reliable strategy for the rapid development of recombinant CHO cell lines.


CHO Site-specific integration CRISPR/Cas9 Cell line development C12orf35 



We would like to thank Dr. Xiangping Zhu, Yuan Gao, and Man Wang at Jecho Biopharmaceuticals Co., Ltd. in Tianjin, China, for professional help in quality analysis of the monoclonal antibodies.


This work was supported in part by the National Natural Science Foundation of China (No. 81273576, 81773621) and the Science & Technology Commission of Shanghai Municipality (No. 15431907000 and 17431904500).

Compliance with ethical standards

Conflict of interest

Authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals experiments.

Supplementary material

253_2018_9021_MOESM1_ESM.pdf (2.9 mb)
ESM 1 (PDF 2995 kb)


  1. Bachu R, Bergareche I, Chasin LA (2015) CRISPR-Cas targeted plasmid integration into mammalian cells via non-homologous end joining. Biotechnol Bioeng 112(10):2154–2162. CrossRefPubMedGoogle Scholar
  2. Baser B, Spehr J, Bussow K, van den Heuvel J (2016) A method for specifically targeting two independent genomic integration sites for co-expression of genes in CHO cells. Methods 95:3–12. CrossRefPubMedGoogle Scholar
  3. Cheng JK, Lewis AM, Kim do S, Dyess T, Alper HS (2016) Identifying and retargeting transcriptional hot spots in the human genome. Biotechnol J 11(8):1100–1109. CrossRefPubMedGoogle Scholar
  4. Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F (2013) Multiplex genome engineering using CRISPR/Cas systems. Science 339(6121):819–823. CrossRefPubMedPubMedCentralGoogle Scholar
  5. Damavandi N, Raigani M, Joudaki A, Davami F, Zeinali S (2017) Rapid characterization of the CHO platform cell line and identification of pseudo attP sites for PhiC31 integrase. Protein Expr Purif 140:60–64. CrossRefPubMedGoogle Scholar
  6. Ding K, Han L, Zong H, Chen J, Zhang B, Zhu J (2017) Production process reproducibility and product quality consistency of transient gene expression in HEK293 cells with anti-PD1 antibody as the model protein. Appl Microbiol Biotechnol 101(5):1889–1898. CrossRefPubMedGoogle Scholar
  7. Doench JG, Fusi N, Sullender M, Hegde M, Vaimberg EW, Donovan KF, Smith I, Tothova Z, Wilen C, Orchard R, Virgin HW, Listgarten J, Root DE (2016) Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nat Biotechnol 34(2):184–191. CrossRefPubMedPubMedCentralGoogle Scholar
  8. Fischer S, Handrick R, Otte K (2015) The art of CHO cell engineering: a comprehensive retrospect and future perspectives. Biotechnol Adv 33(8):1878–1896. CrossRefPubMedGoogle Scholar
  9. Galleguillos SN, Ruckerbauer D, Gerstl MP, Borth N, Hanscho M, Zanghellini J (2017) What can mathematical modelling say about CHO metabolism and protein glycosylation? Comput Struct Biotechnol J 15:212–221. CrossRefPubMedPubMedCentralGoogle Scholar
  10. Gupta SK, Shukla P (2017) Gene editing for cell engineering: trends and applications. Crit Rev Biotechnol 37(5):672–684. CrossRefPubMedGoogle Scholar
  11. He X, Tan C, Wang F, Wang Y, Zhou R, Cui D, You W, Zhao H, Ren J, Feng B (2016) Knock-in of large reporter genes in human cells via CRISPR/Cas9-induced homology-dependent and independent DNA repair. Nucleic Acids Res 44(9):e85. CrossRefPubMedPubMedCentralGoogle Scholar
  12. Hiller GW, Ovalle AM, Gagnon MP, Curran ML, Wang WG (2017) Cell-controlled hybrid perfusion fed-batch CHO cell process provides significant productivity improvement over conventional fed-batch cultures. Biotechnol Bioeng 114(7):1438–1447. CrossRefPubMedGoogle Scholar
  13. Inniss MC, Bandara K, Jusiak B, Lu TK, Weiss R, Wroblewska L, Zhang L (2017) A novel Bxb1 integrase RMCE system for high fidelity site-specific integration of mAb expression cassette in CHO cells. Biotechnol Bioeng 114(8):1837–1846. CrossRefPubMedGoogle Scholar
  14. Irion U, Krauss J, Nusslein-Volhard C (2014) Precise and efficient genome editing in zebrafish using the CRISPR/Cas9 system. Development 141(24):4827–4830. CrossRefPubMedPubMedCentralGoogle Scholar
  15. Jabalameli HR, Zahednasab H, Karimi-Moghaddam A, Jabalameli MR (2015) Zinc finger nuclease technology: advances and obstacles in modelling and treating genetic disorders. Gene 558(1):1–5. CrossRefPubMedGoogle Scholar
  16. Josse L, Xie J, Proud CG, Smales CM (2016) mTORC1 signalling and eIF4E/4E-BP1 translation initiation factor stoichiometry influence recombinant protein productivity from GS-CHOK1 cells. Biochem J 473(24):4651–4664. CrossRefPubMedPubMedCentralGoogle Scholar
  17. Kawabe Y, Shimomura T, Huang S, Imanishi S, Ito A, Kamihira M (2016) Targeted transgene insertion into the CHO cell genome using Cre recombinase-incorporating integrase-defective retroviral vectors. Biotechnol Bioeng 113(7):1600–1610. CrossRefPubMedGoogle Scholar
  18. Kawabe Y, Komatsu S, Komatsu S, Murakami M, Ito A, Sakuma T, Nakamura T, Yamamoto T, Kamihira M (2017) Targeted knock-in of an scFv-Fc antibody gene into the hprt locus of Chinese hamster ovary cells using CRISPR/Cas9 and CRIS-PITCh systems. J Biosci Bioeng.
  19. Kleinstiver BP, Pattanayak V, Prew MS, Tsai SQ, Nguyen NT, Zheng Z, Joung JK (2016) High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects. Nature 529(7587):490–495. CrossRefPubMedPubMedCentralGoogle Scholar
  20. Lai T, Yang Y, Ng SK (2013) Advances in mammalian cell line development technologies for recombinant protein production. Pharmaceuticals (Basel) 6(5):579–603. CrossRefGoogle Scholar
  21. Lee JS, Kallehauge TB, Pedersen LE, Kildegaard HF (2015) Site-specific integration in CHO cells mediated by CRISPR/Cas9 and homology-directed DNA repair pathway. Sci Rep 5:8572. CrossRefPubMedPubMedCentralGoogle Scholar
  22. Lee JS, Grav LM, Pedersen LE, Lee GM, Kildegaard HF (2016) Accelerated homology-directed targeted integration of transgenes in Chinese hamster ovary cells via CRISPR/Cas9 and fluorescent enrichment. Biotechnol Bioeng 113(11):2518–2523. CrossRefPubMedGoogle Scholar
  23. Li S, Gao X, Peng R, Zhang S, Fu W, Zou F (2016) FISH-based analysis of clonally derived CHO cell populations reveals high probability for transgene integration in a terminal region of chromosome 1 (1q13). PLoS One 11(9):e0163893. CrossRefPubMedPubMedCentralGoogle Scholar
  24. Mali P, Aach J, Stranges PB, Esvelt KM, Moosburner M, Kosuri S, Yang L, Church GM (2013) CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nat Biotechnol 31(9):833–838. CrossRefPubMedPubMedCentralGoogle Scholar
  25. Moreno-Mateos MA, Vejnar CE, Beaudoin JD, Fernandez JP, Mis EK, Khokha MK, Giraldez AJ (2015) CRISPRscan: designing highly efficient sgRNAs for CRISPR-Cas9 targeting in vivo. Nat Methods 12(10):982–988. CrossRefPubMedPubMedCentralGoogle Scholar
  26. Nakao H, Harada T, Nakao K, Kiyonari H, Inoue K, Furuta Y, Aiba A (2016) A possible aid in targeted insertion of large DNA elements by CRISPR/Cas in mouse zygotes. Genesis 54(2):65–77. CrossRefPubMedGoogle Scholar
  27. Ran FA, Hsu PD, Lin CY, Gootenberg JS, Konermann S, Trevino AE, Scott DA, Inoue A, Matoba S, Zhang Y, Zhang F (2013) Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell 154(6):1380–1389. CrossRefPubMedPubMedCentralGoogle Scholar
  28. Ritter A, Rauschert T, Oertli M, Piehlmaier D, Mantas P, Kuntzelmann G, Lageyre N, Brannetti B, Voedisch B, Geisse S, Jostock T, Laux H (2016) Disruption of the gene C12orf35 leads to increased productivities in recombinant CHO cell lines. Biotechnol Bioeng 113(11):2433–2442. CrossRefPubMedGoogle Scholar
  29. Sakuma T, Takenaga M, Kawabe Y, Nakamura T, Kamihira M, Yamamoto T (2015) Homologous recombination-independent large gene cassette knock-in in CHO cells using TALEN and MMEJ-directed donor plasmids. Int J Mol Sci 16(10):23849–23866. CrossRefPubMedPubMedCentralGoogle Scholar
  30. Sun T, Li CD, Han L, Jiang H, Xie YQ, Zhang BH, Qian XP, Lu HL, Zhu JW (2015) Functional knockout of FUT8 in Chinese hamster ovary cells using CRISPR/Cas9 to produce a defucosylated antibody. Eng Life Sci 15(6):660–666. CrossRefGoogle Scholar
  31. Templeton N, Lewis A, Dorai H, Qian EA, Campbell MP, Smith KD, Lang SE, Betenbaugh MJ, Young JD (2014) The impact of anti-apoptotic gene Bcl-2 expression on CHO central metabolism. Metab Eng 25:92–102. CrossRefPubMedGoogle Scholar
  32. Wang C, Thudium KB, Han M, Wang XT, Huang H, Feingersh D, Garcia C, Wu Y, Kuhne M, Srinivasan M, Singh S, Wong S, Garner N, Leblanc H, Bunch RT, Blanset D, Selby MJ, Korman AJ (2014) In vitro characterization of the anti-PD-1 antibody nivolumab, BMS-936558, and in vivo toxicology in non-human primates. Cancer Immunol Res 2(9):846–856. CrossRefPubMedGoogle Scholar
  33. Wang X, Kawabe Y, Kato R, Hada T, Ito A, Yamana Y, Kondo M, Kamihira M (2017) Accumulative scFv-Fc antibody gene integration into the hprt chromosomal locus of Chinese hamster ovary cells. J Biosci Bioeng 124(5):583–590. CrossRefPubMedGoogle Scholar
  34. Xu S, Chen H (2016) High-density mammalian cell cultures in stirred-tank bioreactor without external pH control. J Biotechnol 231:149–159. CrossRefPubMedGoogle Scholar
  35. Zhu J (2012) Mammalian cell protein expression for biopharmaceutical production. Biotechnol Adv 30(5):1158–1170. CrossRefPubMedGoogle Scholar
  36. Zhu J (2013) Update on production of recombinant therapeutic protein: transient gene expression. Smithers Rapra Technology Ltd.Google Scholar
  37. Zong H, Han L, Ding K, Wang J, Sun T, Zhang X, Cagliero C, Jiang H, Xie Y, Xu J, Zhang B, Zhu J (2017) Producing defucosylated antibodies with enhanced in vitro antibody-dependent cellular cytotoxicity via FUT8 knockout CHO-S cells. Eng Life Sci 17(7):801–808. CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Engineering Research Center of Cell and Therapeutic Antibody, Ministry of Education, School of PharmacyShanghai Jiao Tong UniversityShanghaiChina
  2. 2.Department of HematologyVU University Medical CenterAmsterdamthe Netherlands
  3. 3.Jecho Laboratories, Inc.FrederickUSA
  4. 4.Instrumental Analysis CenterShanghai Jiao Tong UniversityShanghaiChina

Personalised recommendations