Abstract
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.
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 language | English |
|---|---|
| Publication date | 2017 |
| Number of pages | 1 |
| Publication status | Published - 2017 |
| Event | 25th ESACT Meeting: Cell technologies for innovative therapies - Lausanne, Switzerland Duration: 14 May 2017 → 17 May 2017 Conference number: 25 |
Conference
| Conference | 25th ESACT Meeting |
|---|---|
| Number | 25 |
| Country/Territory | Switzerland |
| City | Lausanne |
| Period | 14/05/2017 → 17/05/2017 |