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Stable Cas9 expression regulates cell growth by facilitating mTORC2 activation

Le Yu1,2, Yi Jin1,2, Jianfeng Chen1,2

  • 1Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, United States.

Nucleic Acids Research
|September 29, 2025
PubMed

Insights

The CRISPR gene-editing tool, Cas9, can unintentionally alter cell growth by activating the mTORC2 pathway through interactions with ribosomal proteins. This discovery highlights the need for safer Cas9 variants in research and therapy.

Area of Science:

  • Molecular Biology
  • Cell Biology
  • Genetics

Background:

  • CRISPR-Cas9 is a powerful gene-editing technology.
  • The impact of Cas9 expression on mammalian cell behavior is not fully understood.
  • Understanding Cas9's off-target effects is crucial for safe applications.

Purpose of the Study:

  • To investigate the effects of stable Cas9 expression on cell growth across various cancer and non-cancerous cell lines.
  • To elucidate the molecular mechanisms underlying Cas9-mediated alterations in cell growth.
  • To identify novel interactions and signaling pathways affected by Cas9.

Main Methods:

  • Systematic profiling of Cas9 expression effects on 32 cell lines.
  • Proteomic analysis to establish the SpCas9 interactome.
  • RNA-sequencing (RNA-seq) to analyze gene expression changes.
  • Biochemical assays to validate protein interactions and pathway activation.

Main Results:

  • Cas9 expression altered cell growth in a subset of tested cell lines.
  • Ribosomal proteins were identified as key interactors of SpCas9.
  • Cas9 expression activated PI3K signaling and enhanced mTORC2 pathway activity.
  • SpCas9 acts as a scaffold, stabilizing the interaction between ribosomal proteins and Sin1, leading to increased mTORC2 activation.

Conclusions:

  • A novel Cas9-ribosome-mTORC2 signaling axis promotes cell growth.
  • Unintended cellular effects of Cas9 need careful consideration in CRISPR applications.
  • Engineering safer Cas9 variants is essential for advancing biomedical research and clinical therapies.

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