Methods for sensitive and high-throughput evaluation of CRISPR RNA-guided nucleases (RGNs) off-targets (OTs) are essential for advancing RGN-based gene therapies. Here we report SURRO-seq for simultaneously evaluating thousands of therapeutic RGN OTs in cells. SURRO-seq captures RGN-induced indels in cells by pooled lentiviral OTs libraries and deep sequencing, an approach comparable and complementary to OTs detection by T7 endonuclease 1, GUIDE-seq, and CIRCLE-seq. Application of SURRO-seq to 8150 OTs from 110 therapeutic RGNs identifies significantly detectable indels in 783 OTs, of which 37 OTs are found in cancer genes and 23 OTs are further validated in five human cell lines by targeted amplicon sequencing. Finally, SURRO-seq reveals that thermodynamically stable wobble base pair (rG•dT) and free binding energy strongly affect RGN specificity. Our study emphasizes the necessity of thoroughly evaluating therapeutic RGN OTs to minimize inevitable off-target effects.

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.

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.

EccDNA is a driver of many cancers and a potential intermediate in other age-related disorders. However, little is known about the mechanisms underlying eccDNA formation in healthy tissue and how aging affects these processes. Here, we present an atlas of eccDNA across seven tissues of male mice spanning four ages. EccDNA correlates with open chromatin characterized by signatures of H3K27ac and H3K4me1. Additionally, the mutational load of eccDNA on genes correlates with tissue-specific transcription and increases logarithmically as a function of transcript level. Still, a population of intron-dense genes with many splice forms remains sheltered from eccDNA formation. We also find that the total number of eccDNA molecules does not increase as mice age, unlike other types of mutations. Our data reveal a link between eccDNA formation and transcript level that may drive gene architecture in mammals.