Stable Cas9 Expression on Cell Growth Regulation Through mTORC2 Activation Facilitation

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 responses and behavior remain unclear. Here, we systematically profiled effects of stable Cas9 expression on growth of 30 cell lines spanning 10 cancer types, observing growth alterations in 12 of them. To uncover the underlying mechanisms, we established the SpCas9 interactome in DU145 and MDA-MB-231 cells, both of which exhibited Cas9-increased growth phenotypes, and identified ribosomal proteins as the top shared interactors. Mechanistic studies revealed that ribosomal proteins such as RPL26 and RPL23a bind Sin1, a key mTORC2 component, relieving Sin1-PH-mediated suppression of the mTOR kinase domain leading to mTORC2 activation. Notably, SpCas9 interacts with both RPL26/RPL23a and Sin1, acting as a "glue" to stabilize their association and enhances 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.