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

Thomas Amann, Anders Holmgaard Hansen, Nusa Pristovsek, Ankita Singh, Gyun Min Lee, Mikael Rørdam Andersen, Helene Faustrup Kildegaard

Research output: Contribution to conferencePosterResearchpeer-review

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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.
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
http://www.esact2017.com/

Conference

Conference25th ESACT Meeting 2017
Number25
CountrySwitzerland
CityLausanne
Period14/05/201717/05/2017
Internet address

Cite this

Amann, T., Hansen, A. H., Pristovsek, N., Singh, A., Min Lee, G., Andersen, M. R., & Kildegaard, H. F. (2017). CHO glyco-engineering using CRISPR/Cas9 multiplexing for protein production with homogeneous N-glycan profiles. Poster session presented at 25th ESACT Meeting 2017, Lausanne, Switzerland.
Amann, Thomas ; Hansen, Anders Holmgaard ; Pristovsek, Nusa ; Singh, Ankita ; Min Lee, Gyun ; Andersen, Mikael Rørdam ; Kildegaard, Helene Faustrup. / CHO glyco-engineering using CRISPR/Cas9 multiplexing for protein production with homogeneous N-glycan profiles. Poster session presented at 25th ESACT Meeting 2017, Lausanne, Switzerland.1 p.
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title = "CHO glyco-engineering using CRISPR/Cas9 multiplexing for protein production with homogeneous N-glycan profiles",
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.",
author = "Thomas Amann and Hansen, {Anders Holmgaard} and Nusa Pristovsek and Ankita Singh and {Min Lee}, Gyun and Andersen, {Mikael R{\o}rdam} and Kildegaard, {Helene Faustrup}",
year = "2017",
language = "English",
note = "25th ESACT Meeting 2017, ESACT 2017 ; Conference date: 14-05-2017 Through 17-05-2017",
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Amann, T, Hansen, AH, Pristovsek, N, Singh, A, Min Lee, G, Andersen, MR & Kildegaard, HF 2017, 'CHO glyco-engineering using CRISPR/Cas9 multiplexing for protein production with homogeneous N-glycan profiles' 25th ESACT Meeting 2017, Lausanne, Switzerland, 14/05/2017 - 17/05/2017, .

CHO glyco-engineering using CRISPR/Cas9 multiplexing for protein production with homogeneous N-glycan profiles. / Amann, Thomas; Hansen, Anders Holmgaard; Pristovsek, Nusa; Singh, Ankita; Min Lee, Gyun; Andersen, Mikael Rørdam; Kildegaard, Helene Faustrup.

2017. Poster session presented at 25th ESACT Meeting 2017, Lausanne, Switzerland.

Research output: Contribution to conferencePosterResearchpeer-review

TY - CONF

T1 - CHO glyco-engineering using CRISPR/Cas9 multiplexing for protein production with homogeneous N-glycan profiles

AU - Amann, Thomas

AU - Hansen, Anders Holmgaard

AU - Pristovsek, Nusa

AU - Singh, Ankita

AU - Min Lee, Gyun

AU - Andersen, Mikael Rørdam

AU - Kildegaard, Helene Faustrup

PY - 2017

Y1 - 2017

N2 - 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.

AB - 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.

M3 - Poster

ER -

Amann T, Hansen AH, Pristovsek N, Singh A, Min Lee G, Andersen MR et al. CHO glyco-engineering using CRISPR/Cas9 multiplexing for protein production with homogeneous N-glycan profiles. 2017. Poster session presented at 25th ESACT Meeting 2017, Lausanne, Switzerland.