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

Kevin Kellner, Ankur Solanki, Thomas Amann, Nga Lao, Niall Barron

Research output: Chapter in Book/Report/Conference proceedingBook chapterResearchpeer-review

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.
Original languageEnglish
Title of host publicationRecombinant Protein Expression in Mammalian Cells
EditorsJohn M. Walker
Volume1850
PublisherHumana Press
Publication date2018
Pages221-235
Chapter15
ISBN (Print)978-1-4939-8729-0
ISBN (Electronic)978-1-4939-8730-6
DOIs
Publication statusPublished - 2018
SeriesMethods in Molecular Biology
ISSN1064-3745

Keywords

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

Cite this

Kellner, K., Solanki, A., Amann, T., Lao, N., & Barron, N. (2018). Targeting miRNAs with CRISPR/Cas9 to Improve Recombinant Protein Production of CHO Cells. In J. M. Walker (Ed.), Recombinant Protein Expression in Mammalian Cells (Vol. 1850, pp. 221-235). Humana Press. Methods in Molecular Biology https://doi.org/10.1007/978-1-4939-8730-6_15
Kellner, Kevin ; Solanki, Ankur ; Amann, Thomas ; Lao, Nga ; Barron, Niall. / Targeting miRNAs with CRISPR/Cas9 to Improve Recombinant Protein Production of CHO Cells. Recombinant Protein Expression in Mammalian Cells . editor / John M. Walker. Vol. 1850 Humana Press, 2018. pp. 221-235 (Methods in Molecular Biology).
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Kellner, K, Solanki, A, Amann, T, Lao, N & Barron, N 2018, Targeting miRNAs with CRISPR/Cas9 to Improve Recombinant Protein Production of CHO Cells. in JM Walker (ed.), Recombinant Protein Expression in Mammalian Cells . vol. 1850, Humana Press, Methods in Molecular Biology, pp. 221-235. https://doi.org/10.1007/978-1-4939-8730-6_15

Targeting miRNAs with CRISPR/Cas9 to Improve Recombinant Protein Production of CHO Cells. / Kellner, Kevin; Solanki, Ankur; Amann, Thomas; Lao, Nga; Barron, Niall.

Recombinant Protein Expression in Mammalian Cells . ed. / John M. Walker. Vol. 1850 Humana Press, 2018. p. 221-235 (Methods in Molecular Biology).

Research output: Chapter in Book/Report/Conference proceedingBook chapterResearchpeer-review

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AB - 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.

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BT - Recombinant Protein Expression in Mammalian Cells

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Kellner K, Solanki A, Amann T, Lao N, Barron N. Targeting miRNAs with CRISPR/Cas9 to Improve Recombinant Protein Production of CHO Cells. In Walker JM, editor, Recombinant Protein Expression in Mammalian Cells . Vol. 1850. Humana Press. 2018. p. 221-235. (Methods in Molecular Biology). https://doi.org/10.1007/978-1-4939-8730-6_15