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Circular DNA can arise from all parts of eukaryotic chromosomes. In yeast, circular ribosomal DNA (rDNA) accumulates dramatically as cells age, however little is known about the accumulation of other chromosome-derived circles or the contribution of such circles to genetic variation in aged cells. We profiled circular DNA in Saccharomyces cerevisiae populations sampled when young and after extensive aging. Young cells possessed highly diverse circular DNA populations but 94% of the circular DNA were lost after ∼15 divisions, whereas rDNA circles underwent massive accumulation to >95% of circular DNA. Circles present in both young and old cells were characterized by replication origins including circles from unique regions of the genome and repetitive regions: rDNA and telomeric Y' regions. We further observed that circles can have flexible inheritance patterns: [HXT6/7circle] normally segregates to mother cells but in low glucose is present in up to 50% of cells, the majority of which must have inherited this circle from their mother. Interestingly, [HXT6/7circle] cells are eventually replaced by cells carrying stable chromosomal HXT6 HXT6/7 HXT7 amplifications, suggesting circular DNAs are intermediates in chromosomal amplifications. In conclusion, the heterogeneity of circular DNA offers flexibility in adaptation, but this heterogeneity is remarkably diminished with age., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Extrachromosomal circular DNA (eccDNA) elements of chromosomal origin are known to be common in a number of eukaryotic species. However, it remains to be addressed whether genomic features such as genome size, the load of repetitive elements within a genome, and/or animal physiology affect the number of eccDNAs. Here, we investigate the distribution and numbers of eccDNAs in a condensed and less repeat-rich genome compared with the human genome, using Columba livia domestica (domestic rock pigeon) as a model organism. By sequencing eccDNA in blood and breast muscle from three pigeon breeds at various ages and with different flight behavior, we characterize 30,000 unique eccDNAs. We identify genomic regions that are likely hotspots for DNA circularization in breast muscle, including genes involved in muscle development. We find that although eccDNA counts do not correlate with the biological age in pigeons, the number of unique eccDNAs in a nonflying breed (king pigeons) is significantly higher (9-fold) than homing pigeons. Furthermore, a comparison between eccDNA from skeletal muscle in pigeons and humans reveals ∼9-10 times more unique eccDNAs per human nucleus. The fraction of eccDNA sequences, derived from repetitive elements, exist in proportions to genome content, that is, human 72.4% (expected 52.5%) and pigeon 8.7% (expected 5.5%). Overall, our results support that eccDNAs are common in pigeons, that the amount of unique eccDNA types per nucleus can differ between species as well as subspecies, and suggest that eccDNAs from repeats are found in proportions relative to the content of repetitive elements in a genome., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Extrachromosomal circular DNA (eccDNA) and ring chromosomes are genetic alterations found in humans with genetic disorders. However, there is a lack of genetic engineering tools to recapitulate and study the biogenesis of eccDNAs. Here, we created a dual-fluorescence biosensor cassette, which upon the delivery of pairs of CRISPR/Cas9 guide RNAs, CRISPR-C, allows us to study the biogenesis of a specific fluorophore expressing eccDNA in human cells. We show that CRISPR-C can generate functional eccDNA, using the novel eccDNA biosensor system. We further reveal that CRISPR-C also can generate eccDNAs from intergenic and genic loci in human embryonic kidney 293T cells and human mammary fibroblasts. EccDNAs mainly forms by end-joining mediated DNA-repair and we show that CRISPR-C is able to generate endogenous eccDNAs in sizes from a few hundred base pairs and ranging up to 207 kb. Even a 47.4 megabase-sized ring chromosome 18 can be created by CRISPR-C. Our study creates a new territory for CRISPR gene editing and highlights CRISPR-C as a useful tool for studying the cellular impact, persistence and function of eccDNAs.

Extrachromosomal circular DNA (eccDNA) derived from chromosomal Ty retrotransposons in yeast can be generated in multiple ways. Ty eccDNA can arise from the circularization of extrachromosomal linear DNA during the transpositional life cycle of retrotransposons, or from circularization of genomic Ty DNA. Circularization may happen through nonhomologous end-joining (NHEJ) of long terminal repeats (LTRs) flanking Ty elements, by Ty autointegration, or by LTR-LTR recombination. By performing an in-depth investigation of sequence reads stemming from Ty eccDNAs obtained from populations of Saccharomyces cerevisiae S288c, we find that eccDNAs predominantly correspond to full-length Ty1 elements. Analyses of sequence junctions reveal no signs of NHEJ or autointegration events. We detect recombination junctions that are consistent with yeast Ty eccDNAs being generated through recombination events within the genome. This opens the possibility that retrotransposable elements could move around in the genome without an RNA intermediate directly through DNA circularization., (Copyright © 2016 by the Genetics Society of America.)