Stable Cas9 expression regulates cell growth by facilitating mTORC2 activation

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.