Generative Modelling of Oncogene-carrying Extrachromosomal Circular DNA Biogenesis and Dynamics in Cells

Abstract

ABSTRACTExtrachromosomal circular DNAs (ecDNA) are focal gene amplifications frequently associated with cancer development and often indicating a poor prognosis. To understand the early dynamics of oncogene-carrying ecDNAs, we previously developed CRISPR-C, a tool for precise ecDNA generation by deleting specific chromosomal regions. Here, we adapted CRISPR-C to recreate tumor ecDNAs. This method also allowed us to enhance ecDNA generation efficiency by directly delivering Cas9 protein and sgRNAs as a ribonucleoprotein complex. By using the modified CRISPR-C, we successfully generated ecDNAs carrying oncogenes (EGFR, CDK4, MDM2, MYC, MYCN, FGFR2, ABCB1,andDHFR) in various human cell types. Furthermore, we demonstrated that our method could generate chimeric ecDNAs composed of target sequences from distant intra or inter-chromosomal regions. Using these generative ecDNA cell models, we studied the oncogene ecDNA expression and stability. TheMDM2expression was increased after CRISPR-C, whileCDK4was decreased indicating genomic-context dependent effect. The copy number of CRISPR-C generatedCDK4was ecDNA increased in cells after a long period of treatment with theCDK4inhibitor palbociclib. Unlike CDK4, the CRISPR-C generatedABCB1ecDNA was unstable in cells under normal growth conditions, but is stably retained when the cells were treated with colcemid, a recognized substrate for ABCB1. We thus provide valuable tools and an attractive platform for studying ecDNA biogenesisy and in vitro drug screening on ecDNA stability.