Selective degradation of oncogenic extrachromosomal DNA by type I-E CRISPR

Abstract

Extrachromosomal DNA (ecDNA) is a major driver of oncogene amplification, intratumoral heterogeneity and therapy resistance across multiple cancer types, yet there are currently no effective strategies to selectively eliminate it. Here, we show that the type I-E CRISPR system can specifically target and degrade ecDNA in human cancer cell lines (COLO320DM and GBM39). By designing guide RNAs targeting ecDNA-specific breakpoints absent from chromosomal DNA, we achieved efficient depletion of MYC- and EGFR-containing ecDNAs in COLO320DM and GBM39 cells. Loss of ecDNA was accompanied by diminished oncogene signaling, disrupted ecDNA architecture, and impaired tumor cell proliferation, without detectable chromosomal off-target activity. These findings establish a proof-of-concept framework for directly targeting oncogenic ecDNAs and highlight type I-E CRISPR as a promising platform for therapeutic development in ecDNA-driven cancers.