biostudies-literatureUnknown53(18)Yu LMary Kay Ash FoundationUniversity of North CarolinaChapel Hill University Cancer ResearchClustered 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 RPL2Nucleic acids researchgkaf965https://www.ebi.ac.uk/biostudies/studies/S-EPMC12477591biostudies-literatureStable Cas9 expression regulates cell growth by facilitating mTORC2 activation.PMC12477591Yu LSu SCloer EWZhu ZChen JMajor MBGongora JLiu PJin YWilkerson EMfalseStable 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 RPL22025-01-01T00:00:00Z2025 Sep2026-06-04T01:46:12.647Z2026-05-04T03:13:42.638ZS-EPMC124775914102050110.1093/nar/gkaf965