Stable Cas9 expression regulates cell growth by facilitating mTORC2 activation

Clustered regularly interspaced short palindromic repeats (CRISPR), widely used for gene editing, relies on bacterial endonucleases like Cas9 to study gene functions and develop therapies. However, its potential effects on mammalian cellular behavior remain unclear. Here, we systematically profiled effects of stable Cas9 expression on growth of 32 cell lines spanning 9 cancer types and non-cancerous cells, finding growth alterations in a subset. To investigate mechanisms, we established the SpCas9 interactome in DU145 and MDA-MB-231 cells, both showing Cas9-enhanced growth, and identified ribosomal proteins as the top shared interactors. RNA-seq analysis revealed that Cas9 expression in DU145 cells activated PI3K signaling. Mechanistic studies showed that ribosomal proteins, including RPL26 and RPL23a, bind to Sin1, a core mTORC2 component, leading to mTORC2 activation. Notably, SpCas9 interacts with both RPL26/RPL23a and Sin1, acting as a scaffold to stabilize their association and enhance mTORC2 activation, even in the absence of growth factors. Our study systematically characterizes Cas9’s effects on cell growth regulation and uncovers a novel Cas9-ribosome-mTORC2 signaling axis that promotes cell growth. These findings underscore the need to consider unintended cellular effects in CRISPR applications and highlight the importance of engineering safer Cas9 variants for biomedical research and clinical therapies.