Modelling the generation of oncogene-carrying extrachromosomal circular DNA through CRISPR-C

State of the art sequencing methods opened broad perspectives for exploring the landscape and varied structure of extrachromosomal circular DNA (eccDNA), a prominent form of focal gene amplification frequently associated with the development of cancer and malignancy. However, the vast amount of data and emerging theories resulting from these sequencing efforts have underscored the urgent need for a reliable approach to model the generation and amplification of recurrent oncogene‐carrying eccDNAs. In our laboratory, we have devised a molecular tool called CRISPR‐C, which enables precise generation of eccDNA by deleting specific chromosomal regions. To enhance the efficacy of eccDNA generation, we have adapted CRISPR‐C by directly delivering the Cas9 enzyme and two editing sgRNAs as a ribonucleoprotein (RNP) complex. Leveraging our eccDNA‐biosensor cell model, which allows us to scrutinize the biogenesis of a particular fluorophore‐encoding eccDNA in human cells, we have observed a substantial 6‐7 fold increase in eccDNA generation efficiency. Employing the modified CRISPR‐C, we have successfully generated eccDNAs harbouring oncogenes (e.g., EGFR, CDK4, MDM2, or DHFR) across a spectrum of cell types, including HEK‐293T, U2OS osteosarcoma, MCF7 breast cancer cells, primary human astrocytes, and skin fibroblasts. By employing DNA Fluorescent In Situ Hybridization, we visualized deletion of the targeted chromosomal region and have ascertained the extracellular presence or chromosomal relocation of the eccDNA. Furthermore, we have demonstrated that our system is capable of generation of chimeric eccDNAs composed of target sequences from distant intra or inter‐chromosomal regions. Upon induction of CDK4 and MDM2‐carrying eccDNA formation, we have observed diminished viability and proliferation capacity of U2OS cells one‐week post‐procedure, with recovery apparent after three weeks. This effect has corresponded with a global downregulation in transcriptional activity of genes located within the CRISPR‐C target regions across most experimental groups, subsequently restored after an additional two weeks, thus indicating prevalent gene deletions at that specific time point. By integrating the refined version of CRISPR‐C with our eccDNA biosensor system, we have elucidated the impact of suppressing various DNA repair and DNA sensing‐related genes on eccDNA generation. Our findings highlight the significant but non‐exclusive role of DNA‐PKCs, the principal kinase involved in non‐homologous end‐joining, in eccDNA biogenesis. Additionally, we have discovered the involvement of DNA ligase 4 in the reintegration of the generated DNA fragment, while intriguingly not in the circularization of the eccDNA.