Site-specific integration in CHO cells mediated by CRISPR/Cas9 and homology-directed DNA repair pathway

Jae Seong Lee; Thomas Beuchert Kallehauge; Lasse Ebdrup Pedersen; Helene Faustrup Kildegaard

doi:10.1038/srep08572

Site-specific integration in CHO cells mediated by CRISPR/Cas9 and homology-directed DNA repair pathway

Jae Seong Lee, Thomas Beuchert Kallehauge, Lasse Ebdrup Pedersen, Helene Faustrup Kildegaard

Novo Nordisk Foundation Center for Biosustainability

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Abstract

Chinese hamster ovary (CHO) cells are the most widely used mammalian hosts for production of therapeutic proteins. However, development of recombinant CHO cell lines has been hampered by unstable and variable transgene expression caused by random integration. Here we demonstrate efficient targeted gene integration into site-specific loci in CHO cells using CRISPR/Cas9 genome editing system and compatible donor plasmid harboring a gene of interest (GOI) and short homology arms. This strategy has enabled precise insertion of a 3.7-kb gene expression cassette at defined loci in CHO cells following a simple drug-selection, resulting in homogeneous transgene expression. Taken together, the results displayed here can help pave the way for the targeting of GOI to specific loci in CHO cells, increasing the likelihood of generating isogenic cell lines with consistent protein production.

Original language	English
Article number	8572
Journal	Scientific Reports
Volume	5
Number of pages	11
ISSN	2045-2322
DOIs	https://doi.org/10.1038/srep08572
Publication status	Published - 2015

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abstract = "Chinese hamster ovary (CHO) cells are the most widely used mammalian hosts for production of therapeutic proteins. However, development of recombinant CHO cell lines has been hampered by unstable and variable transgene expression caused by random integration. Here we demonstrate efficient targeted gene integration into site-specific loci in CHO cells using CRISPR/Cas9 genome editing system and compatible donor plasmid harboring a gene of interest (GOI) and short homology arms. This strategy has enabled precise insertion of a 3.7-kb gene expression cassette at defined loci in CHO cells following a simple drug-selection, resulting in homogeneous transgene expression. Taken together, the results displayed here can help pave the way for the targeting of GOI to specific loci in CHO cells, increasing the likelihood of generating isogenic cell lines with consistent protein production.",

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AU - Kildegaard, Helene Faustrup

N1 - This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission fromthe license holder in order to reproduce thematerial. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

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AB - Chinese hamster ovary (CHO) cells are the most widely used mammalian hosts for production of therapeutic proteins. However, development of recombinant CHO cell lines has been hampered by unstable and variable transgene expression caused by random integration. Here we demonstrate efficient targeted gene integration into site-specific loci in CHO cells using CRISPR/Cas9 genome editing system and compatible donor plasmid harboring a gene of interest (GOI) and short homology arms. This strategy has enabled precise insertion of a 3.7-kb gene expression cassette at defined loci in CHO cells following a simple drug-selection, resulting in homogeneous transgene expression. Taken together, the results displayed here can help pave the way for the targeting of GOI to specific loci in CHO cells, increasing the likelihood of generating isogenic cell lines with consistent protein production.

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Site-specific integration in CHO cells mediated by CRISPR/Cas9 and homology-directed DNA repair pathway

Abstract

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