Beyond chromosomes: exploring the diverse functions of extrachromosomal circular DNA

Extrachromosomal circular DNA (eccDNA) is defined as a form of circular DNA that is widespread across various biological contexts and exists independently of chromosomal structures. The current distribution of total eccDNA identified in both healthy and tumor tissues shows genomic coverage ranging from 1.18% to 30.73%. EccDNAs are double-stranded, covalently closed, circular DNA molecules that originate from mechanisms such as somatic rearrangements, genomic instability, and hypertranscription. EccDNA possesses an open chromatin structure, which can participate in transcriptional regulation across the entire genome and mediate extensive interactions between eccDNAs and between eccDNA and chromosomes. EccDNAs vary from a few dozen base pairs to megabases (Mb) and may include anything from small non-coding sequences to entire genes and regulatory elements. Current research methods limit the ability to knock down or knock out eccDNA like genomic DNA. Overexpressing individual eccDNAs is also challenging, making it difficult to explore their molecular function. This review examines the underexplored potential functions of eccDNA, with particular emphasis on its role as an enhancer and its involvement in the production of transcribed mRNAs and non-coding RNAs (ncRNAs). In addition, we provided a method for in vitro synthesis of eccDNA and a method for generating eccDNA in cells based on CRISPR/Cas9 technology, which provides a research foundation for elucidating its biological functions and mechanisms of action. Finally, this paper presents innovative prospects from a forward-looking perspective regarding the functional mechanisms and potential applications of eccDNA in various biological scenarios.