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Peer-reviewedCC BYFrontiers in bioengineering and biotechnology

Same hydrolase knockouts, different outcomes: CRISPR/Cas9 editing workflow shapes resulting cellular phenotype and transcriptome in CHO

  • Linus Weiß1
  • Stefan Schneider1
  • Kerstin Otte1
  • Simon Fischer2
  1. 1Institute of Applied Biotechnology, Biberach University of Applied Sciences , ,
  2. 2Cell Line Development, Boehringer Ingelheim , ,
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Abstract

CRISPR/Cas9-based multi-gene knockout strategies are widely applied to engineer Chinese hamster ovary (CHO) cells, yet the impact of editing workflow on cellular outcomes remains poorly understood. Here, we systematically compare sequential and multiplex genome editing approaches to generate CHO cell lines lacking nine polysorbate (PS)-degrading hydrolases. Both strategies achieved comparable functional outcomes, markedly improving PS stability. However, they produced distinct phenotypic and transcriptomic trajectories. Sequential editing led to progressive declines in growth and viability, increased cell size and DNA content, and extensive transcriptomic remodeling. In contrast, multiplex editing resulted in milder phenotypic alterations and a substantially smaller transcriptional footprint, albeit with increased clone-to-clone heterogeneity. Transcriptome analyses revealed limited overlap between final states, indicating workflow-specific adaptation despite the same intended knockout set. Importantly, transcriptional changes were primarily associated with clonal selection and iterative manipulation rather than reflecting a shared response to the targeted knockouts themselves. These findings demonstrate that genome editing workflows can shape cellular phenotypes independently of intended genetic modifications. For cell line development, multiplex editing offers a strategy to reduce development time and limit undesirable drift when targeting non-essential genes, highlighting the need to consider engineering strategy as a critical design parameter.

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Cite

BibTeXRIS
BibTeX
@article{weiß2026same,
  title = {{Same hydrolase knockouts, different outcomes: CRISPR/Cas9 editing workflow shapes resulting cellular phenotype and transcriptome in CHO}},
  author = {Linus Weiß and Stefan Schneider and Kerstin Otte and Simon Fischer},
  journal = {Frontiers in bioengineering and biotechnology},
  year = {2026},
  month = oct,
  doi = {10.3389/fbioe.2026.1923843},
  url = {https://europepmc.org/article/PMC/PMC13724508},
  note = {CC BY},
}
RIS
TY  - JOUR
TI  - Same hydrolase knockouts, different outcomes: CRISPR/Cas9 editing workflow shapes resulting cellular phenotype and transcriptome in CHO
AU  - Linus Weiß
AU  - Stefan Schneider
AU  - Kerstin Otte
AU  - Simon Fischer
PY  - 2026
DA  - 2026/10/19
JO  - Frontiers in bioengineering and biotechnology
DO  - 10.3389/fbioe.2026.1923843
UR  - https://europepmc.org/article/PMC/PMC13724508
AB  - CRISPR/Cas9-based multi-gene knockout strategies are widely applied to engineer Chinese hamster ovary (CHO) cells, yet the impact of editing workflow on cellular outcomes remains poorly understood. Here, we systematically compare sequential and multiplex genome editing approaches to generate CHO cell lines lacking nine polysorbate (PS)-degrading hydrolases. Both strategies achieved comparable functional outcomes, markedly improving PS stability. However, they produced distinct phenotypic and transcriptomic trajectories. Sequential editing led to progressive declines in growth and viability, increased cell size and DNA content, and extensive transcriptomic remodeling. In contrast, multiplex editing resulted in milder phenotypic alterations and a substantially smaller transcriptional footprint, albeit with increased clone-to-clone heterogeneity. Transcriptome analyses revealed limited overlap between final states, indicating workflow-specific adaptation despite the same intended knockout set. Importantly, transcriptional changes were primarily associated with clonal selection and iterative manipulation rather than reflecting a shared response to the targeted knockouts themselves. These findings demonstrate that genome editing workflows can shape cellular phenotypes independently of intended genetic modifications. For cell line development, multiplex editing offers a strategy to reduce development time and limit undesirable drift when targeting non-essential genes, highlighting the need to consider engineering strategy as a critical design parameter.
SN  - 2296-4185
ER  -