Conference paper
CHO glyco-engineering using CRISPR/Cas9 multiplexing for protein production with homogeneous N-glycan profiles
Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark1
CHO Cell Line Engineering and Design, Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark2
CHO in Silico Engineering of Glycosylation and Protein Quality (CiSe), Research Groups, Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark3
CHO Core, Translational Management, Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark4
iLoop, Translational Management, Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark5
Department of Biotechnology and Biomedicine, Technical University of Denmark6
Network Engineering of Eukaryotic Cell factories, Section for Synthetic Biology, Department of Biotechnology and Biomedicine, Technical University of Denmark7
Combining the chinese hamster ovary (CHO) - K1 draft genome1,2, identified CHO glycosyltransferases3 and the power of multiplexing gene knock-outs with CRISPR/Cas94 via co-transfection of Cas9 and one single guiding RNA (sgRNA) per target, we generated 20 Rituximab expressing CHO-S cell lines differing in amount and combination of insertions or deletions (indels) in the targeted genes.
Clones harboring 9, 6 and 4 indels were further investigated for growth, Rituximab productivity and secretome N-glycosylation. This resulted in clones with prolonged viabilites, no changes in N-glycan galactose contents but an increase of matured and sialylated N-glycan structures in the secretome. Additionally we point out, that multiplexing an increasing amount of genes most likely results in clones only revealing a few of all possible combinations of the targets and is highly driven by the sgRNA efficiency which can differ from each other by factor 4, even after FACS sorting.
Language: | English |
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Year: | 2017 |
Proceedings: | 25th ESACT Meeting |
Types: | Conference paper |
ORCIDs: | Amann, Thomas , Hansen, Anders Holmgaard , Pristovsek, Nusa , Singh, Ankita , Andersen, Mikael Rørdam and Kildegaard, Helene Faustrup |