CRISPR-Cas9 induced formation of Myc-containing ecDNA drives carcinogenesis in mouse liver and cancer phenotypes in human cell models

Over 40% of human cancers harbor extrachromosomal DNA (ecDNA), which correlates with therapy resistance and poor prognosis. Yet research is limited by a lack of robust de novo ecDNA models. We present a CRISPR-Cas9 based approach for generation of ecDNA in cell lines and mouse liver. In HEK293 cells, CRISPR-induced MYC-bearing ecDNA ([MYCcircle]) promoted cancer hallmarks, including enhanced proliferation, increased migration, and reduced apoptosis. To model ecDNA in mice, we combined Cas9 knock in mice with lipid-nanoparticle delivery of sgRNAs in the liver targeting the Myc–Pvt1 locus. When combined with Trp53 or Pten loss, the resultant [Myccircle] caused hepatocellular carcinomas with amplified levels of [Myccircle] and elevated, heterogenous Myc expression, supporting evidence of ecDNA involvement in tumorigenesis. Our approach offers a fast and versatile tool, where target-sequence flexibility allows rapid generation of animal and cell models with any investigator-specified ecDNA, thereby enabling study of the direct role of ecDNA in cancer progression.