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Targeting miRNAs with CRISPR/Cas9 to Improve Recombinant Protein Production of CHO Cells

  • Kevin KellnerEmail author
  • Ankur Solanki
  • Thomas Amann
  • Nga Lao
  • Niall Barron
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1850)

Abstract

MicroRNAs with their unique ability to target hundreds of genes have been highlighted as powerful tools to improve bioprocess behavior of cells. The common approaches to stably deplete miRNAs are the use of sponge decoy transcripts or shRNA inhibitors, which requires the introduction and expression of extra genetic material. As an alternative, we implemented the CRISPR/Cas9 system in our laboratory to generate Chinese hamster ovary (CHO) cells which lack the expression of a specific miRNA for the purpose of functional studies. To implement the system, miR-27a/b was chosen as it has been shown to be upregulated during hypothermic conditions and therefore may be involved in controlling CHO cell growth and recombinant protein productivity. In this chapter, we present a protocol for targeting miRNAs in CHO cells using CRISPR/Cas9 and the analysis of the resulting phenotype, using miR-27 as an example. We showed that it is possible to target miRNAs in CHO cells and achieved ≥80% targeting efficiency. Indel analysis and TOPO-TA cloning combined with Sanger sequencing showed a range of different indels. Furthermore, it was possible to identify clones with no detectable expression of mature miR-27b. Depletion of miR-27b led to improved viability in late stages of batch and fed-batch cultures making it a potentially interesting target to improve bioprocess performance of CHO cells.

Key words

CRISPR/Cas9 MicroRNA depletion Chinese hamster ovary cells Cell line engineering Productivity 

Notes

Acknowledgments

The authors would like to recognise the support of Science Foundation Ireland IvP award no. 13/IA/1963 (KK, AS, NL and NB) and Horizon 2020 Marie-S-Curie ITN www.echo-systems.eu (TA).

References

  1. 1.
    Baek E, Noh SM, Lee GM (2017) Anti-apoptosis engineering for improved protein production from CHO cells. Methods Mol Biol 1603:71–85.  https://doi.org/10.1007/978-1-4939-6972-2_5CrossRefPubMedGoogle Scholar
  2. 2.
    Le Fourn V, Girod PA, Buceta M, Regamey A, Mermod N (2014) CHO cell engineering to prevent polypeptide aggregation and improve therapeutic protein secretion. Metab Eng 21:91–102.  https://doi.org/10.1016/j.ymben.2012.12.003CrossRefPubMedGoogle Scholar
  3. 3.
    Josse L, Smales CM, Tuite MF (2012) Engineering the chaperone network of CHO cells for optimal recombinant protein production and authenticity. Methods Mol Biol 824:595–608.  https://doi.org/10.1007/978-1-61779-433-9_32CrossRefPubMedGoogle Scholar
  4. 4.
    Toussaint C, Henry O, Durocher Y (2016) Metabolic engineering of CHO cells to alter lactate metabolism during fed-batch cultures. J Biotechnol 217:122–131.  https://doi.org/10.1016/j.jbiotec.2015.11.010CrossRefPubMedGoogle Scholar
  5. 5.
    Wang Q, Yin B, Chung CY, Betenbaugh MJ (2017) Glycoengineering of CHO cells to improve product quality. Methods Mol Biol 1603:25–44.  https://doi.org/10.1007/978-1-4939-6972-2_2CrossRefPubMedGoogle Scholar
  6. 6.
    Barron N, Sanchez N, Kelly P, Clynes M (2011) MicroRNAs: tiny targets for engineering CHO cell phenotypes? Biotechnol Lett 33(1):11–21.  https://doi.org/10.1007/s10529-010-0415-5CrossRefPubMedGoogle Scholar
  7. 7.
    Jadhav V, Hackl M, Druz A, Shridhar S, Chung CY, Heffner KM, Kreil DP, Betenbaugh M, Shiloach J, Barron N, Grillari J, Borth N (2013) CHO microRNA engineering is growing up: recent successes and future challenges. Biotechnol Adv 31(8):1501–1513.  https://doi.org/10.1016/j.biotechadv.2013.07.007CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Fromm B, Billipp T, Peck LE, Johansen M, Tarver JE, King BL, Newcomb JM, Sempere LF, Flatmark K, Hovig E, Peterson KJ (2015) A uniform system for the annotation of vertebrate microRNA genes and the evolution of the human microRNAome. Annu Rev Genet 49:213–242.  https://doi.org/10.1146/annurev-genet-120213-092023CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Pasquinelli AE (2012) MicroRNAs and their targets: recognition, regulation and an emerging reciprocal relationship. Nat Rev Genet 13(4):271–282.  https://doi.org/10.1038/nrg3162CrossRefPubMedGoogle Scholar
  10. 10.
    Gammell P, Barron N, Kumar N, Clynes M (2007) Initial identification of low temperature and culture stage induction of miRNA expression in suspension CHO-K1 cells. J Biotechnol 130(3):213–218 S0168-1656(07)00289-1 [pii]CrossRefGoogle Scholar
  11. 11.
    Agrawal R, Pandey P, Jha P, Dwivedi V, Sarkar C, Kulshreshtha R (2014) Hypoxic signature of microRNAs in glioblastoma: insights from small RNA deep sequencing. BMC Genomics 15:686.  https://doi.org/10.1186/1471-2164-15-686CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Chhabra R, Adlakha YK, Hariharan M, Scaria V, Saini N (2009) Upregulation of miR-23a-27a-24-2 cluster induces caspase-dependent and -independent apoptosis in human embryonic kidney cells. PLoS One 4(6):e5848.  https://doi.org/10.1371/journal.pone.0005848CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Mertens-Talcott SU, Chintharlapalli S, Li X, Safe S (2007) The oncogenic microRNA-27a targets genes that regulate specificity protein transcription factors and the G2-M checkpoint in MDA-MB-231 breast cancer cells. Cancer Res 67(22):11001–11011 67/22/11001 [pii]CrossRefGoogle Scholar
  14. 14.
    Kluiver J, Gibcus JH, Hettinga C, Adema A, Richter MK, Halsema N, Slezak-Prochazka I, Ding Y, Kroesen BJ, van den Berg A (2012) Rapid generation of microRNA sponges for microRNA inhibition. PLoS One 7(1):e29275.  https://doi.org/10.1371/journal.pone.0029275CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Kelly PS (2014) Enhancing CHO cell productivity through the stable depletion of microRNA-23. Dublin City University, DublinGoogle Scholar
  16. 16.
    Sanchez N, Kelly P, Gallaghe C, Lao NT, Clarke C, Clynes M, Barron N (2014) CHO cell culture longevity and recombinant protein yield are enhanced by depletion of miR-7 activity via sponge decoy vectors. Biotechnol J 9(3):396–404.  https://doi.org/10.1002/biot.201300325CrossRefPubMedGoogle Scholar
  17. 17.
    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.  https://doi.org/10.1038/srep08572CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Ronda C, Pedersen LE, Hansen HG, Kallehauge TB, Betenbaugh MJ, Nielsen AT, Kildegaard HF (2014) Accelerating genome editing in CHO cells using CRISPR Cas9 and CRISPy, a web-based target finding tool. Biotechnol Bioeng 111(8):1604–1616.  https://doi.org/10.1002/bit.25233CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Chang H, Yi B, Ma R, Zhang X, Zhao H, Xi Y (2016) CRISPR/cas9, a novel genomic tool to knock down microRNA in vitro and in vivo. Sci Rep 6.  https://doi.org/10.1038/srep22312
  20. 20.
    Zhao Y, Dai Z, Liang Y, Yin M, Ma K, He M, Ouyang H, Teng CB (2014) Sequence-specific inhibition of microRNA via CRISPR/CRISPRi system. Sci Rep 4:3943.  https://doi.org/10.1038/srep03943CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Griffith A, Kelly P, Vencken S, Lao N, Greene M, Clynes M, Barron N (2017) miR-CATCH identifies biologically active miRNA regulators of the pro-survival gene XIAP in Chinese hamster ovary cells. J Biotechnol.  https://doi.org/10.1002/biot201700299
  22. 22.
    Clarke C, Doolan P, Barron N, Meleady P, O'Sullivan F, Gammell P, Melville M, Leonard M, Clynes M (2011) Predicting cell-specific productivity from CHO gene expression. J Biotechnol 151:159–165.  https://doi.org/10.1016/j.jbiotec.2010.11.016CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Kevin Kellner
    • 1
    Email author
  • Ankur Solanki
    • 1
  • Thomas Amann
    • 2
  • Nga Lao
    • 1
  • Niall Barron
    • 3
    • 4
  1. 1.National Institute for Cellular BiotechnologyDublin City UniversityDublinIreland
  2. 2.Novo Nordisk Foundation Center for BiosustainabilityTechnical University of DenmarkKgs. LyngbyDenmark
  3. 3.National Institute for Bioprocessing Research and TrainingDublinIreland
  4. 4.School of Chemical and Bioprocess EngineeringUniversity College DublinDublinIreland

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