CHO glyco-engineering using CRISPR/Cas9 multiplexing for protein production with homogeneous N-glycan profiles

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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.
Original languageEnglish
Publication date2017
Number of pages1
Publication statusPublished - 2017
Event25th ESACT Meeting 2017 - Lausanne, Switzerland
Duration: 14 May 201717 May 2017
Conference number: 25


Conference25th ESACT Meeting 2017
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