Microfluidic isolation of extrachromosomal circular DNA through selective digestion of plasmids and linear DNA using immobilized nucleases.

Extrachromosomal circular DNA (eccDNA) refers to small circular DNA molecules that are distinct from chromosomal DNA and play diverse roles in various biological processes. They are also explored as potential biomarkers for disease diagnosis and precision medicine. However, isolating eccDNA from tissues and plasma is challenging due to low abundance and the presence of interfering linear DNA, requiring time-consuming processes and expert handling. Our study addresses this by utilizing a microfluidic chip tailored for eccDNA isolation, leveraging microfluidic principles for enzymatic removal of non-circular DNA. Our approach involves integrating restriction enzymes into the microfluidic chip, enabling selective digestion of mitochondrial and linear DNA fragments while preserving eccDNA integrity. This integration is facilitated by an in situ photo-polymerized emulsion inside microchannels, creating a porous monolithic structure suitable for immobilizing restriction and exonuclease enzymes (restriction enzyme MssI and exonuclease ExoV). Evaluation using control DNA mixtures and plasma samples with artificially introduced eccDNA demonstrated that our microfluidic chips reduce linear DNA by over 99%, performing comparable to conventional off-chip methods but with substantially faster digestion times, allowing for a remarkable 76-fold acceleration in overall sample preparation time. This technological advancement holds great promise for enhancing the isolation and analysis of eccDNA from tissue and plasma and the potential for increasing the speed of other molecular methods with multiple enzymatic steps.

Extrachromosomal Circular DNA: An Emerging Potential Biomarker for Inflammatory Bowel Diseases?

Inflammatory bowel disease (IBD) comprising ulcerative colitis and Crohn's disease is a chronic immune-mediated disease which affects the gastrointestinal tract with a relapsing and remitting course, causing lifelong morbidity. IBD pathogenesis is determined by multiple factors including genetics, immune and microbial factors, and environmental factors. Although therapy options are expanding, remission rates are unsatisfiable, and together with the disease course, response to therapy remains unpredictable. Therefore, the identification of biomarkers that are predictive for the disease course and response to therapy is a significant challenge. Extrachromosomal circular DNA (eccDNA) fragments exist in all tissue tested so far. These fragments, ranging in length from a few hundreds of base pairs to mega base pairs, have recently gained more interest due to technological advances. Until now, eccDNA has mainly been studied in relation to cancer due to its ability to act as an amplification site for oncogenes and drug resistance genes. However, eccDNA could also play an important role in inflammation, expressed both locally in the- involved tissue and at distant sites. Here, we review the current evidence on the molecular mechanisms of eccDNA and its role in inflammation and IBD. Additionally, the potential of eccDNA as a tissue or plasma marker for disease severity and/or response to therapy is evaluated.

Variation of extrachromosomal circular DNA in cancer cell lines.

The presence of oncogene carrying eccDNAs is strongly associated with carcinogenesis and poor patient survival. Tumour biopsies and in vitro cancer cell lines are frequently utilized as models to investigate the role of eccDNA in cancer. However, eccDNAs are often lost during the in vitro growth of cancer cell lines, questioning the reproducibility of studies utilizing cancer cell line models. Here, we conducted a comprehensive analysis of eccDNA variability in seven cancer cell lines (MCA3D, PDV, HaCa4, CarC, MIA-PaCa-2, AsPC-1, and PC-3). We compared the content of unique eccDNAs between triplicates of each cell line and found that the number of unique eccDNA is specific to each cell line, while the eccDNA sequence content varied greatly among triplicates (∼ 0-1% eccDNA coordinate commonality). In the PC-3 cell line, we found that the large eccDNA (ecDNA) with MYC is present in high-copy number in an NCI cell line isolate but not present in ATCC isolates. Together, these results reveal that the sequence content of eccDNA is highly variable in cancer cell lines. This highlights the importance of testing cancer cell lines before use, and to enrich for subclones in cell lines with the desired eccDNA to get relatively pure population for studying the role of eccDNA in cancer.

Methods for the purification and detection of single nucleotide KRAS mutations on extrachromosomal circular DNA in human plasma.

BACKGROUNDS: Despite recent advances, many cancers are still detected too late for curative treatment. There is, therefore, a need for the development of new diagnostic methods and biomarkers. One approach may arise from the detection of extrachromosomal circular DNA (eccDNA), which is part of cell-free DNA in human plasma. AIMS: First, we assessed and compared two methods for the purification of eccDNA from plasma. Second, we tested for an easy diagnostic application of eccDNA liquid biopsy-based assays. MATERIALS & METHODS: For the comparison we tested a solid-phase silica purification method and a phenol/chloroform method with salt precipitation. For the diagnostic application of eccDNA we developed and tested a qPCR primer-based SNP detection system, for the detection of two well-established cancer-causing KRAS mutations (G12V and G12R) on circular DNA. This investigation was supported by purifying, sequencing, and analysing clinical plasma samples for eccDNAs containing KRAS mutant alleles in 0.5 mL plasma from 16 pancreatic ductal adenocarcinoma patients and 19 healthy controls. RESULTS: In our method comparison we observed, that following exonuclease treatment a lower eccDNA yield was found for the phenol/chloroform method (15.7%-26.7%) compared with the solid-phase purification approach (47.8%-65.9%). For the diagnostic application of eccDNA tests, the sensitivity of the tested qPCR assay only reached ~10-3 in a background of 105 wild type (wt) KRAS circular entities, which was not improved by general amplification or primer-based inhibition of wt KRAS amplification. Furthermore, we did not detect eccDNA containing KRAS in any of the clinical samples. DISCUSSION: A potential explanation for our inability to detect any KRAS mutations in the clinical samples may be related to the general low abundance of eccDNA in plasma. CONCLUSION: Taken together our results provide a benchmark for eccDNA purification methods while raising the question of what is required for the optimal fast and sensitive detection of SNP mutations on eccDNA with greater sensitivity than primer-based qPCR detection.

Circular and Circulating DNA in Inflammatory Bowel Disease: From Pathogenesis to Potential Molecular Therapies.

Inflammatory bowel diseases (IBD), including Crohn's Disease (CD) and Ulcerative Colitis (UC) are chronic multifactorial disorders which affect the gastrointestinal tract with variable extent. Despite extensive research, their etiology and exact pathogenesis are still unknown. Cell-free DNAs (cfDNAs) are defined as any DNA fragments which are free from the origin cell and able to circulate into the bloodstream with or without microvescicles. CfDNAs are now being increasingly studied in different human diseases, like cancer or inflammatory diseases. However, to date it is unclear how IBD etiology is linked to cfDNAs in plasma. Extrachromosomal circular DNA (eccDNA) are non-plasmidic, nuclear, circular and closed DNA molecules found in all eukaryotes tested. CfDNAs appear to play an important role in autoimmune diseases, inflammatory processes, and cancer; recently, interest has also grown in IBD, and their role in the pathogenesis of IBD has been suggested. We now suggest that eccDNAs also play a role in IBD. In this review, we have comprehensively collected available knowledge in literature regarding cfDNA, eccDNA, and structures involving them such as neutrophil extracellular traps and exosomes, and their role in IBD. Finally, we focused on old and novel potential molecular therapies and drug delivery systems, such as nanoparticles, for IBD treatment.

Did circular DNA shape the evolution of mammalian genomes?

Extrachromosomal circular DNA (eccDNA) can shape the genomes of somatic cells, but how it impacts genomes across generations is largely unexplored. We propose that genomes can rearrange via circular intermediates across generations and show that up to 6% of a mammalian genome can have changed gene order through eccDNA.

CReSIL: accurate identification of extrachromosomal circular DNA from long-read sequences.

Extrachromosomal circular DNA (eccDNA) of chromosomal origin is found in many eukaryotic species and cell types, including cancer, where eccDNAs with oncogenes drive tumorigenesis. Most studies of eccDNA employ short-read sequencing for their identification. However, short-read sequencing cannot resolve the complexity of genomic repeats, which can lead to missing eccDNA products. Long-read sequencing technologies provide an alternative to constructing complete eccDNA maps. We present a software suite, Construction-based Rolling-circle-amplification for eccDNA Sequence Identification and Location (CReSIL), to identify and characterize eccDNA from long-read sequences. CReSIL's performance in identifying eccDNA, with a minimum F1 score of 0.98, is superior to the other bioinformatic tools based on simulated data. CReSIL provides many useful features for genomic annotation, which can be used to infer eccDNA function and Circos visualization for eccDNA architecture investigation. We demonstrated CReSIL's capability in several long-read sequencing datasets, including datasets enriched for eccDNA and whole genome datasets from cells containing large eccDNA products. In conclusion, the CReSIL suite software is a versatile tool for investigating complex and simple eccDNA in eukaryotic cells.

Targeted removal of mitochondrial DNA from mouse and human extrachromosomal circular DNA with CRISPR-Cas9.

Extrachromosomal circular DNA (eccDNA) of chromosomal origin is common in eukaryotic cells. Amplification of oncogenes on large eccDNA (ecDNA) can drive biological processes such as tumorigenesis, and identification of eccDNA by sequencing after removal of chromosomal DNA is therefore important for understanding their impact on the expressed phenotype. However, the circular mitochondrial DNA (mtDNA) might challenge the detection of eccDNA because the average somatic cell has hundreds of copies of mtDNA. Here we show that 61.2-99.5% of reads from eccDNA-enriched samples correspond to mtDNA in mouse tissues. We have developed a method to selectively remove mtDNA from total circular DNA by CRISPR/Cas9 guided cleavage of mtDNA with one single-guide RNA (sgRNA) or two sgRNAs followed by exonuclease degradation of the linearized mtDNA. Sequencing revealed that mtDNA reads were 85.9% ± 12.6% removed from eccDNA of 9 investigated mouse tissues. CRISPR/Cas9 cleavage also efficiently removed mtDNA from a human HeLa cell line and colorectal cancer samples. We identified up to 14 times more, and also larger eccDNA in CRISPR/Cas9 treated colorectal cancer samples than in untreated samples. We foresee that the method can be applied to effectively remove mtDNA from any eukaryotic species to obtain higher eccDNA yields.

Circle-Seq reveals genomic and disease-specific hallmarks in urinary cell-free extrachromosomal circular DNAs.

BACKGROUND: Extrachromosomal circular deoxyribonucleic acid (eccDNA) is evolving as a valuable biomarker, while little is known about its presence in urine. METHODS: Here, we report the discovery and analysis of urinary cell-free eccDNAs (ucf-eccDNAs) in healthy controls and patients with advanced chronic kidney disease (CKD) by Circle-Seq. RESULTS: Millions of unique ucf-eccDNAs were identified and comprehensively characterised. The ucf-eccDNAs are GC-rich. Most ucf-eccDNAs are less than 1000 bp and are enriched in four pronounced peaks at 207, 358, 553 and 732 bp. Analysis of the genomic distribution of ucf-eccDNAs shows that eccDNAs are found on all chromosomes but enriched on chromosomes 17, 19 and 20 with a high density of protein-coding genes, CpG islands, short interspersed transposable elements (SINEs) and simple repeat elements. Analysis of eccDNA junction sequences further suggests that microhomology and palindromic repeats might be involved in eccDNA formation. The ucf-eccDNAs in CKD patients are significantly higher than those in healthy controls. Moreover, eccDNA with miRNA genes is highly enriched in CKD ucf-eccDNA. CONCLUSIONS: This work discovers and provides the first deep characterisation of ucf-eccDNAs and suggests ucf-eccDNA as a valuable noninnvasive biomarker for urogenital disorder diagnosis and monitoring.

A unifying model for extrachromosomal circular DNA load in eukaryotic cells.

Extrachromosomal circular DNA (eccDNA) with exons and whole genes are common features of eukaryotic cells. Work from especially tumours and the yeast Saccharomyces cerevisiae has revealed that eccDNA can provide large selective advantages and disadvantages. Besides the phenotypic effect due to expression of an eccDNA fragment, eccDNA is different from other mutations in that it is released from 1:1 segregation during cell division. This means that eccDNA can quickly change copy number, pickup secondary mutations and reintegrate into a chromosome to establish substantial genetic variation that could not have evolved via canonical mechanisms. We propose a unifying 5-factor model for conceptualizing the eccDNA load of a eukaryotic cell, emphasizing formation, replication, segregation, selection and elimination. We suggest that the magnitude of these sequential events and their interactions determine the copy number of eccDNA in mitotically dividing cells. We believe that our model will provide a coherent framework for eccDNA research, to understand its biology and the factors that can be manipulated to modulate eccDNA load in eukaryotic cells.

Extrachromosomal circular DNA in cancer: history, current knowledge, and methods.

Extrachromosomal circular DNA (eccDNA) is a closed-circle, nuclear, nonplasmid DNA molecule found in all tested eukaryotes. eccDNA plays important roles in cancer pathogenesis, evolution of tumor heterogeneity, and therapeutic resistance. It is known under many names, including very large cancer-specific circular extrachromosomal DNA (ecDNA), which carries oncogenes and is often amplified in cancer cells. Our understanding of eccDNA has historically been limited and fragmented. To provide better a context of new and previous research on eccDNA, in this review we give an overview of the various names given to eccDNA at different times. We describe the different mechanisms for formation of eccDNA and the methods used to study eccDNA thus far. Finally, we explore the potential clinical value of eccDNA.

Circular DNA in the human germline and its association with recombination.

Extrachromosomal circular DNA (eccDNA) is common in somatic tissue, but its existence and effects in the human germline are unexplored. We used microscopy, long-read DNA sequencing, and new analytic methods to document thousands of eccDNAs from human sperm. EccDNAs derived from all genomic regions and mostly contained a single DNA fragment, although some consisted of multiple fragments. The generation of eccDNA inversely correlates with the meiotic recombination rate, and chromosomes with high coding-gene density and Alu element abundance form the least eccDNA. Analysis of insertions in human genomes further indicates that eccDNA can persist in the human germline when the circular molecules reinsert themselves into the chromosomes. Our results suggest that eccDNA has transient and permanent effects on the germline. They explain how differences in the physical and genetic map might arise and offer an explanation of how Alu elements coevolved with genes to protect genome integrity against deleterious mutations producing eccDNA.

Isolation, characterization, and genome assembly of Barnettozyma botsteinii sp. nov. and novel strains of Kurtzmaniella quercitrusa isolated from the intestinal tract of the termite Macrotermes bellicosus.

Bioconversion of hemicelluloses into simpler sugars leads to the production of a significant amount of pentose sugars, such as d-xylose. However, efficient utilization of pentoses by conventional yeast production strains remains challenging. Wild yeast strains can provide new industrially relevant characteristics and efficiently utilize pentose sugars. To explore this strategy, we isolated gut-residing yeasts from the termite Macrotermes bellicosus collected in Comoé National Park, Côte d'Ivoire. The yeasts were classified through their Internal Transcribed Spacer/Large Subunit sequence, and their genomes were sequenced and annotated. We identified a novel yeast species, which we name Barnettozyma botsteinii sp. nov. 1118T (MycoBank: 833563, CBS 16679T and IBT 710) and two new strains of Kurtzmaniella quercitrusa: var. comoensis (CBS 16678, IBT 709) and var. filamentosus (CBS 16680, IBT 711). The two K. quercitrusa strains grow 15% faster on synthetic glucose medium than Saccharomyces cerevisiae CEN.PKT in acidic conditions (pH = 3.2) and both strains grow on d-xylose as the sole carbon source at a rate of 0.35 h-1. At neutral pH, the yeast form of K. quercitrusa var. filamentosus, but not var. comoensis, switched to filamentous growth in a carbon source-dependent manner. Their genomes are 11.0-13.2 Mb in size and contain between 4888 and 5475 predicted genes. Together with closely related species, we did not find any relationship between gene content and ability to grow on xylose. Besides its metabolism, K. quercitrusa var. filamentosus has a large potential as a production organism, because of its capacity to grow at low pH and to undergo a dimorphic shift.

Replicative aging is associated with loss of genetic heterogeneity from extrachromosomal circular DNA in Saccharomyces cerevisiae.

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.

Near-Random Distribution of Chromosome-Derived Circular DNA in the Condensed Genome of Pigeons and the Larger, More Repeat-Rich Human Genome.

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.

Sensitive detection of circular DNAs at single-nucleotide resolution using guided realignment of partially aligned reads.

BACKGROUND: Circular DNA has recently been identified across different species including human normal and cancerous tissue, but short-read mappers are unable to align many of the reads crossing circle junctions hence limiting their detection from short-read sequencing data. RESULTS: Here, we propose a new method, Circle-Map that guides the realignment of partially aligned reads using information from discordantly mapped reads to map the short unaligned portions using a probabilistic model. We compared Circle-Map to similar up-to-date methods for circular DNA and RNA detection and we demonstrate how the approach implemented in Circle-Map dramatically increases sensitivity for detection of circular DNA on both simulated and real data while retaining high precision. CONCLUSION: Circle-Map is an easy-to-use command line tool that implements the required pipeline to accurately detect circular DNA from circle enriched next generation sequencing experiments. Circle-Map is implemented in python3.6 and it is freely available at https://github.com/iprada/Circle-Map.

To Be or Not to Be: Circular RNAs or mRNAs From Circular DNAs?

In recent years, there has been a growing interest in circular RNAs (circRNAs) since they are involved in a wide spectrum of cellular functions that might have a large impact on phenotype and disease. CircRNAs are mainly recorded by RNA-Seq and computational methods focused on the detection of back-splicing junction sequences considered the diagnostic feature of circRNAs. While some protocols remove linear RNA prior to sequencing, many have characterized circRNAs by sorting through total RNA sequencing data without excluding the possibility that some linear RNA can provide the same signal as a circRNA. Recent studies have revealed that circular DNAs of chromosomal origin are common in eukaryotic genomes and that they can be transcribed. Transcription events across the junction of circular DNAs would result in a transcript with a junction similar to those present in circRNAs. Therefore, in this report, we want to draw attention to transcripts from such circular DNAs both as an interesting new player in the transcriptome and also as a confounding factor that must be taken into account when studying circRNAs.

RNAi as a Tool to Study Virulence in the Pathogenic Yeast Candida glabrata.

The yeast Candida glabrata is a major opportunistic pathogen causing mucosal and systemic infections in humans. Systemic infections caused by this yeast have high mortality rates and are difficult to treat due to this yeast's intrinsic and frequently adapting antifungal resistance. To understand and treat C. glabrata infections, it is essential to investigate the molecular basis of C. glabrata virulence and resistance. We established an RNA interference (RNAi) system in C. glabrata by expressing the Dicer and Argonaute genes from Saccharomyces castellii (a budding yeast with natural RNAi). Our experiments with reporter genes and putative virulence genes showed that the introduction of RNAi resulted in 30 and 70% gene-knockdown for the construct-types antisense and hairpin, respectively. The resulting C. glabrata RNAi strain was used for the screening of a gene library for new virulence-related genes. Phenotypic profiling with a high-resolution quantification of growth identified genes involved in the maintenance of cell integrity, antifungal drugs, and ROS resistance. The genes identified by this approach are promising targets for the treatment of C. glabrata infections.

Lifelong physical activity is associated with promoter hypomethylation of genes involved in metabolism, myogenesis, contractile properties and oxidative stress resistance in aged human skeletal muscle.

Lifelong regular physical activity is associated with reduced risk of type 2 diabetes (T2D), maintenance of muscle mass and increased metabolic capacity. However, little is known about epigenetic mechanisms that might contribute to these beneficial effects in aged individuals. We investigated the effect of lifelong physical activity on global DNA methylation patterns in skeletal muscle of healthy aged men, who had either performed regular exercise or remained sedentary their entire lives (average age 62 years). DNA methylation was significantly lower in 714 promoters of the physically active than inactive men while methylation of introns, exons and CpG islands was similar in the two groups. Promoters for genes encoding critical insulin-responsive enzymes in glycogen metabolism, glycolysis and TCA cycle were hypomethylated in active relative to inactive men. Hypomethylation was also found in promoters of myosin light chain, dystrophin, actin polymerization, PAK regulatory genes and oxidative stress response genes. A cluster of genes regulated by GSK3ß-TCF7L2 also displayed promoter hypomethylation. Together, our results suggest that lifelong physical activity is associated with DNA methylation patterns that potentially allow for increased insulin sensitivity and a higher expression of genes in energy metabolism, myogenesis, contractile properties and oxidative stress resistance in skeletal muscle of aged individuals.