Optimized CRISPR-Cas9 system for efficient engineering of ecDNA in cancer cells.

ABSTRACT: Extrachromosomal DNA (ecDNA) amplification represents an emerging mechanism underlying oncogene amplification, tumor heterogeneity, and drug resistance in cancer. However, the biology of ecDNA remains poorly understood because tools to engineer ecDNAs and precisely monitor their dynamics are limited. In particular, genome engineering strategies have not been established for ecDNA, which exists in tens to hundreds of copies within a single cell. Here, we report a systematic validation of ecDNA editing using standard CRISPR-Cas9 system and optimized CRISPR-Cas9 system with safeguard single-guide RNAs (sgRNAs), in which the addition of cytosine extensions finely reduces excessive Cas9 activity. The conventional CRISPR-Cas9 system induced severe cytotoxicity and markedly reduced ecDNA copy num

SUBMITTER: Sugimoto Y 

PROVIDER: S-EPMC12802944 | biostudies-literature | 2026 Jan

REPOSITORIES: biostudies-literature