Extrachromosomal circular DNAs (eccDNAs) carrying random genomic segments are broadly found across different cancer types, but their molecular functions and impact in gastric cancer (GC) are rarely known. In this study, we aimed to investigate the potential role of eccDNA in GC. Using the Circle-seq strategy, we observed the eccDNA abundance in gastric cancer tissues (GCT) was aberrantly higher than that of normal adjacent tissues (NAT). The high abundance of eccDNAs carrying oncogene-segments in GCT may represent the DNA damage products of amplified oncogenes. Analysis of GCT over-represented eccDNA carrying enhancer (eccEnhancer) based on data from FANTOM5 project combined with TCGA database suggested the GC over-represented eccEnhancers may contribute to development of GC. GC over-represented eccDNAs carrying pre-miRNA (eccMIR) were enriched to multiple cancer-relevant signal pathways by KEGG analysis. We then synthesized the top six GC over-represented eccMIRs and found four of them enabled high expression of miRNAs and down-regulation of miRNA-target genes in MGC803 cells. Furthermore, we observed the inheritance of GC over-represented eccMIRs benefited host cell proliferation and promoted the aggressive features of host cells. Altogether, this study revealed the GC over-represented eccDNAs carrying functional genomic segments were related to the carcinogenesis of GC and presented the capability to facilitate cancer progression, suggesting the cancerous eccDNAs may serve as a dynamic reservoir for genome plasticity and rapid adaptive evolution of cancer. Therefore, blocking the pathways for eccDNAs generation may provide a novel therapeutic strategy for the treatment of gastric cancer.

Extrachromosomal DNA (ecDNA) presents a promising target for cancer therapy; however, its spatial–temporal diversity and influence on tumor evolution and the immune microenvironment remain largely unclear. We apply computational methods to analyze ecDNA from whole-genome sequencing data of 595 patients with urothelial carcinoma. We demonstrate that ecDNA drives clonal evolution through structural rearrangements during malignant transformation and recurrence of urothelial carcinoma. This supports a model wherein tumors evolve via the selective expansion of ecDNA-bearing cells. Through multiregional sampling of tumors, we demonstrate that ecDNA contributes to the evolution of multifocality and increased intratumoral heterogeneity. ecDNA is present in 36% of urothelial carcinoma tumors and correlates with an immunosuppressive phenotype and poor prognosis. Single-cell RNA sequencing analyses reveal that ecDNA+ malignant cells exhibit diminished expression of MHC class I molecules, enabling them to evade T-cell immunity. Finally, we show that sequencing of urinary sediment–derived DNA has excellent specificity in detecting ecDNA. Significance: Our comprehensive analysis of ecDNA in urothelial carcinoma reveals its crucial role in driving the evolution and heterogeneity of multifocal cancer, as well as its early involvement in tumorigenesis. Moreover, this study sheds light on immune evasion mechanisms associated with ecDNA and offers valuable insights for developing targeted therapeutic strategies.

CRISPR/Cas9 is an adaptive immunity system in bacteria and most archaea ( Koonin and Makarova, 2009; Horvath and Barrangou, 2010). The CRISPR/Cas9 gene editing system is comprised of two key components, a small guide RNA (gRNA) and a Cas9 endonuclease ( Deltcheva et al., 2011; Jinek et al., 2012). The gRNA is a chimeric RNA molecule of tracrRNA and crRNA ( Ran et al., 2013) which guides the Cas9 protein to the target site in the genome. Therefore, selecting a target site with high on-target activity and low off-target effect is crucial for gene editing. Previously, we have discovered that the gene editing activity of CRISPR-Cas9 in mammalian cells was affected by several factors, such as the secondary structure and chromatin accessibility of the guide sequences ( Jensen et al., 2017). Strikingly, previous results from us and other research groups consistently revealed that the CRISPR gRNA activities are highly variable. Thus, several in silico gRNA design web tools and algorithms have been developed to facilitate CRISPR design and applications.

Extrachromosomal circular DNAs (eccDNAs) are a type of circular DNAs originating from but independent of chromosomal DNAs. Nowadays, with the rapid development of sequencing and bioinformatics, the accuracy of eccDNAs detection has significantly improved. This advancement has consequently enhanced the feasibility of exploring the biological characteristics and functions of eccDNAs. This review elucidates the potential mechanisms of eccDNA generation, the existing methods for their detection and analysis, and their basic features. Furthermore, it focuses on the biological functions of eccDNAs in regulating gene expression under both physiological and pathological conditions. Additionally, the review summarizes the clinical implications of eccDNAs in human cancers and health.

CRISPR gene therapy is one promising approach for treatment of Duchenne muscular dystrophy (DMD), which is caused by a large spectrum of mutations in the dystrophin gene. To broaden CRISPR gene editing strategies for DMD treatment, we report the efficient restoration of dystrophin expression in induced myotubes by SpCas9 and dual guide RNAs (gRNAs). We first sequenced 32 deletion junctions generated by this editing method and revealed that non-homologous blunt-end joining represents the major indel type. Based on this predictive repair outcome, efficient in-frame deletion of a part of DMD exon 51 was achieved in HEK293T cells with plasmids expressing SpCas9 and dual gRNAs. More importantly, we further corrected a frameshift mutation in human DMD (exon45del) fibroblasts with SpCas9-dual gRNA ribonucleoproteins. The edited DMD fibroblasts were transdifferentiated into myotubes by lentiviral-mediated overexpression of a human MYOD transcription factor. Restoration of DMD expression at both the mRNA and protein levels was confirmed in the induced myotubes. With further development, the combination of SpCas9-dual gRNA-corrected DMD patient fibroblasts and transdifferentiation may provide a valuable therapeutic strategy for DMD.

Background: Extrachromosomal circular DNA (eccDNA) has emerged as a promising biomarker for disease diagnosis and prognosis prediction. However, its role in type 2 diabetes remains unexplored.Objective: To investigate the characteristics and dynamics of circulating eccDNAs in newly diagnosed type 2 diabetes mellitus (T2DM) patients undergoing short‐term intensive insulin therapy (SIIT), a highly effective treatment for inducing long‐term glycemic remission.Methods: We conducted Circle‐Seq analysis on plasma samples from 35 T2DM patients at three time points: pre‐SIIT, post‐SIIT, and 1‐year post‐SIIT. Our analysis encompassed the characterization of eccDNA features, including GC content, eccDNA length distribution, genomic distribution, and the genes in eccDNAs.Results: Following SIIT, we observed an increase in plasma eccDNA load, suggesting metabolic alterations during therapy. Notably, a correlation was identified between eccDNA profiles and glycemia in T2DM, both quantitatively and genetically. Our analysis also revealed the frequent presence of metabolism‐related genes within T2DM plasma eccDNAs, some of which spanned gene exons and/or fractions.Conclusion: This study represents the first report of cell‐free eccDNA in T2DM and underscores a compelling association between cell‐free eccDNA and profound glycemic changes. These findings highlight the potential of eccDNAs as crucial players in the context of T2DM and glycemic control.