One-step generation of triple knockout CHO cell lines using CRISPR/Cas9 and fluorescent enrichment

Grav, Lise Marie; Lee, Jae Seong; Gerling, Signe; Kallehauge, Thomas Beuchert; Hansen, Anders Holmgaard; Kol, Stefan; Lee, Gyun Min; Pedersen, Lasse Ebdrup; Kildegaard, Helene Faustrup

doi:10.1002/biot.201500027

Cited by 131CRISPR and Genetic EngineeringViral Infectious Diseases and Gene Expression in InsectsAnimal Genetics and Reproduction

One-step generation of triple knockout CHO cell lines using CRISPR/Cas9 and fluorescent enrichment

Grav, Lise Marie, Lee, Jae Seong, Gerling, Signe, Kallehauge, Thomas Beuchert, Hansen, Anders Holmgaard, Kol, Stefan, Lee, Gyun Min, Pedersen, Lasse Ebdrup +1 more·2015·Biotechnology Journal

DOI 10.1002/biot.201500027

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Abstract

The CRISPR/Cas9 genome editing technology has previously been shown to be a highly efficient tool for generating gene disruptions in CHO cells. In this study we further demonstrate the applicability and efficiency of CRISPR/Cas9 genome editing by disrupting FUT8, BAK and BAX simultaneously in a multiplexing setup in CHO cells. To isolate Cas9‐expressing cells from transfected cell pools, GFP was linked to the Cas9 nuclease via a 2A peptide. With this method, the average indel frequencies generated at the three genomic loci were increased from 11% before enrichment to 68% after enrichment. Despite the high number of genome editing events in the enriched cell pools, no significant off‐target effects were observed from off‐target prediction followed by deep sequencing. Single cell sorting of enriched multiplexed cells and deep sequencing of 97 clones revealed the presence of four single, 23 double and 34 triple gene‐disrupted cell lines. Further characterization of selected potential triple knockout clones confirmed the removal of Bak and Bax protein and disrupted fucosylation activity as expected. The knockout cell lines showed improved resistance to apoptosis compared to wild‐type CHO‐S cells. Taken together, multiplexing with CRISPR/Cas9 can accelerate genome engineering efforts in CHO cells even further.

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CHO cells where simultaneous disruption of FUT8, BAX and BAK gene by CRISPR/Cas9 was achieved and confirmed by deep sequencing showed increased resistance to apoptosis.
“As an example, CHO cells, where simultaneous disruption of FUT8, BAX and BAK gene by CRISP/Cas9 was achieved and confirmed by deep sequencing, showed increased resistance to apoptosis (Grav et al., 2015).”
From Therapeutic glycoprotein production in mammalian cells (2017) · cites this paper as Grav et al., 2015

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Authors: Grav, L. M., Lee, J. S., Gerling, S., Kallehauge, T. B., Hansen, A. H., Kol, S., Lee, G. M., Pedersen, L. E., & Kildegaard, H. F.
Affiliations: Novo Nordisk Foundation (DK)
Korea Advanced Institute of Science and Technology (KR)
Corresponding author: Kildegaard, Helene Faustrup
ORCID: Grav, Lise Marie
Lee, Jae Seong
Kallehauge, Thomas Beuchert
Hansen, Anders Holmgaard
Kol, Stefan
Lee, Gyun Min
Pedersen, Lasse Ebdrup
Kildegaard, Helene Faustrup
Venue: Biotechnology Journal
Publisher: Wiley
Published: 2015-04-30
Volume: 10
Issue: 9
Pages: 1446-1456
ISSN: 1860-6768 (ISSN-L)
1860-7314
DOI: 10.1002/biot.201500027
OpenAlex: W2069942860
Cited by: 131
References: 29
Citations by year: 2026: 7 · 2025: 8 · 2024: 6 · 2023: 5 · 2022: 12 · 2021: 13 · 2020: 11 · 2019: 18 · 2018: 18 · 2017: 15 · 2016: 10 · 2015: 7 · 2013: 1
FWCI: 7.23
Type: journal article
Language: en
Open access: closed
Licence: onlinelibrary.wiley.com/termsAndConditions#vor (vor)
Locations: Biotechnology Journal (publishedVersion)
PubMed (publishedVersion)
Technical University of Denmark, DTU Orbit (Technical University of Denmark, DTU) (submittedVersion)
Funders: The Novo Nordisk Foundation
Topics: Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Viral Infectious Diseases and Gene Expression in Insects
Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Animal Genetics and Reproduction
Keywords: CRISPR/Cas9 genome editing, CHO cells, multiplex genome editing, 2A peptide, FUT8, Bak, Bax, apoptosis resistance, fucosylation, off-target effects, single-cell sorting
MeSH terms: Animals, Biotechnology, Cell Survival, Cricetulus, Flow Cytometry, Cricetinae, CHO Cells, Apoptosis, RNA Editing, Green Fluorescent Proteins, Gene Knockout Techniques, CRISPR-Cas Systems

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One-step generation of triple knockout CHO cell lines using CRISPR/Cas9 and fluorescent enrichment

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