Enhancing stability of recombinant CHO cells by CRISPR/Cas9-mediated site-specific integration into regions with distinct histone modifications
Chinese hamster ovary (CHO) cells are the most important platform for producing biotherapeutics. Random integration of a transgene into epigenetically instable regions of the genome results in silencing of the gene of interest and loss of productivity during upstream processing. Therefore, cost- and...
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2022-10-01
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| Series: | Frontiers in Bioengineering and Biotechnology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fbioe.2022.1010719/full |
| _version_ | 1811240946293014528 |
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| author | Oliver Hertel Oliver Hertel Anne Neuss Tobias Busche David Brandt Jörn Kalinowski Janina Bahnemann Thomas Noll Thomas Noll |
| author_facet | Oliver Hertel Oliver Hertel Anne Neuss Tobias Busche David Brandt Jörn Kalinowski Janina Bahnemann Thomas Noll Thomas Noll |
| author_sort | Oliver Hertel |
| collection | DOAJ |
| description | Chinese hamster ovary (CHO) cells are the most important platform for producing biotherapeutics. Random integration of a transgene into epigenetically instable regions of the genome results in silencing of the gene of interest and loss of productivity during upstream processing. Therefore, cost- and time-intensive long-term stability studies must be performed. Site-specific integration into safe harbors is a strategy to overcome these limitations of conventional cell line design. Recent publications predict safe harbors in CHO cells based on omics data sets or by learning from random integrations, but those predictions remain theory. In this study, we established a CRISPR/Cas9-mediated site-specific integration strategy based on ChIP-seq data to improve stability of recombinant CHO cells. Therefore, a ChIP experiment from the exponential and stationary growth phase of a fed-batch cultivation of CHO-K1 cells yielded 709 potentially stable integration sites. The reporter gene eGFP was integrated into three regions harboring specific modifications by CRISPR/Cas9. Targeted Cas9 nanopore sequencing showed site-specific integration in all 3 cell pools with a specificity between 23 and 73%. Subsequently, the cells with the three different integration sites were compared with the randomly integrated donor vector in terms of transcript level, productivity, gene copy numbers and stability. All site-specific integrations showed an increase in productivity and transcript levels of up to 7.4-fold. In a long-term cultivation over 70 generations, two of the site-specific integrations showed a stable productivity (>70%) independent of selection pressure. |
| first_indexed | 2024-04-12T13:29:32Z |
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| institution | Directory Open Access Journal |
| issn | 2296-4185 |
| language | English |
| last_indexed | 2024-04-12T13:29:32Z |
| publishDate | 2022-10-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Bioengineering and Biotechnology |
| spelling | doaj.art-955939bd790248a7a7791dd84a7eb3482022-12-22T03:31:14ZengFrontiers Media S.A.Frontiers in Bioengineering and Biotechnology2296-41852022-10-011010.3389/fbioe.2022.10107191010719Enhancing stability of recombinant CHO cells by CRISPR/Cas9-mediated site-specific integration into regions with distinct histone modificationsOliver Hertel0Oliver Hertel1Anne Neuss2Tobias Busche3David Brandt4Jörn Kalinowski5Janina Bahnemann6Thomas Noll7Thomas Noll8Cell Culture Technology, Faculty of Technology, Bielefeld University, Bielefeld, GermanyCenter for Biotechnology (CeBiTec), Bielefeld University, Bielefeld, GermanyBiochemical Engineering (AVT.BioVT), RWTH Aachen University, Aachen, GermanyCenter for Biotechnology (CeBiTec), Bielefeld University, Bielefeld, GermanyCenter for Biotechnology (CeBiTec), Bielefeld University, Bielefeld, GermanyCenter for Biotechnology (CeBiTec), Bielefeld University, Bielefeld, GermanyInstitute of Physics, University of Augsburg, Augsburg, GermanyCell Culture Technology, Faculty of Technology, Bielefeld University, Bielefeld, GermanyCenter for Biotechnology (CeBiTec), Bielefeld University, Bielefeld, GermanyChinese hamster ovary (CHO) cells are the most important platform for producing biotherapeutics. Random integration of a transgene into epigenetically instable regions of the genome results in silencing of the gene of interest and loss of productivity during upstream processing. Therefore, cost- and time-intensive long-term stability studies must be performed. Site-specific integration into safe harbors is a strategy to overcome these limitations of conventional cell line design. Recent publications predict safe harbors in CHO cells based on omics data sets or by learning from random integrations, but those predictions remain theory. In this study, we established a CRISPR/Cas9-mediated site-specific integration strategy based on ChIP-seq data to improve stability of recombinant CHO cells. Therefore, a ChIP experiment from the exponential and stationary growth phase of a fed-batch cultivation of CHO-K1 cells yielded 709 potentially stable integration sites. The reporter gene eGFP was integrated into three regions harboring specific modifications by CRISPR/Cas9. Targeted Cas9 nanopore sequencing showed site-specific integration in all 3 cell pools with a specificity between 23 and 73%. Subsequently, the cells with the three different integration sites were compared with the randomly integrated donor vector in terms of transcript level, productivity, gene copy numbers and stability. All site-specific integrations showed an increase in productivity and transcript levels of up to 7.4-fold. In a long-term cultivation over 70 generations, two of the site-specific integrations showed a stable productivity (>70%) independent of selection pressure.https://www.frontiersin.org/articles/10.3389/fbioe.2022.1010719/fullCRISPR/Cas9CHOcell line developmentChIP-seqepigeneticssafe harbor |
| spellingShingle | Oliver Hertel Oliver Hertel Anne Neuss Tobias Busche David Brandt Jörn Kalinowski Janina Bahnemann Thomas Noll Thomas Noll Enhancing stability of recombinant CHO cells by CRISPR/Cas9-mediated site-specific integration into regions with distinct histone modifications Frontiers in Bioengineering and Biotechnology CRISPR/Cas9 CHO cell line development ChIP-seq epigenetics safe harbor |
| title | Enhancing stability of recombinant CHO cells by CRISPR/Cas9-mediated site-specific integration into regions with distinct histone modifications |
| title_full | Enhancing stability of recombinant CHO cells by CRISPR/Cas9-mediated site-specific integration into regions with distinct histone modifications |
| title_fullStr | Enhancing stability of recombinant CHO cells by CRISPR/Cas9-mediated site-specific integration into regions with distinct histone modifications |
| title_full_unstemmed | Enhancing stability of recombinant CHO cells by CRISPR/Cas9-mediated site-specific integration into regions with distinct histone modifications |
| title_short | Enhancing stability of recombinant CHO cells by CRISPR/Cas9-mediated site-specific integration into regions with distinct histone modifications |
| title_sort | enhancing stability of recombinant cho cells by crispr cas9 mediated site specific integration into regions with distinct histone modifications |
| topic | CRISPR/Cas9 CHO cell line development ChIP-seq epigenetics safe harbor |
| url | https://www.frontiersin.org/articles/10.3389/fbioe.2022.1010719/full |
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