Metabolic rewiring plays a crucial role in the energy metabolism of hepatocytes during steatosis. However, the precise alterations in energy metabolism remain unclear. The aim of this study was to investigate the effects of lipid exposure on cellular energy metabolism and gene expression in a cellular model of non-alcoholic fatty liver disease (NAFLD). We induced hepatocyte steatosis in the hepatocyte cell line Huh7 via treatment with high levels of palmitate and oleate for 24 h. We then analysed transcriptomics, proteomics, mitochondrial phenotypes, and cellular energy metabolism. Fatty acid-treated cells presented significant accumulation of lipid droplets and reduced viability. Real-time bioenergetics analyses demonstrated a shift towards mitochondrial respiration for energy production, accompanied by a reduction in glycolytic adenosine triphosphate (ATP) production. However, the overall rate of ATP production remained constant. RNA-seq analysis revealed altered expression of 149 transcripts associated with lipid storage and catabolism, whereas 172 proteins presented significantly altered levels and were enriched in RNA processing and splicing functions. Two genes, ACAA2 and PLIN2, were significantly altered at both the transcript and protein levels and may be crucial for maintaining mitochondrial function in early non-alcoholic fatty liver (NAFL). Our NAFLD model demonstrated that the reprogramming of genes involved in lipid storage and catabolism is crucial for early NAFL pathogenesis development. This study offers new insights with valuable implications for designing future research into novel therapeutic strategies.

Despite representing approximately 30

Design of guide RNA (gRNA) with high efficiency and specificity is vital for successful application of the CRISPR gene editing technology. Although many machine learning (ML) and deep learning (DL)-based tools have been developed to predict gRNA activities, a systematic and unbiased evaluation of their predictive performance is still needed. Here, we provide a brief overview of in silico tools for CRISPR design and assess the CRISPR datasets and statistical metrics used for evaluating model performance. We benchmark seven ML and DL-based CRISPR-Cas9 editing efficiency prediction tools across nine CRISPR datasets covering six cell types and three species. The DL models CRISPRon and DeepHF outperform the other models exhibiting greater accuracy and higher Spearman correlation coefficient across multiple datasets. We compile all CRISPR datasets and in silico prediction tools into a GuideNet resource web portal, aiming to facilitate and streamline the sharing of CRISPR datasets. Furthermore, we summarize features affecting CRISPR gene editing activity, providing important insights into model performance and the further development of more accurate CRISPR prediction models.

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 urothelial carcinoma (UC) patients. We demonstrate that ecDNA drives clonal evolution through structural rearrangements during malignant transformation and recurrence of UC. This supports a model wherein tumors evolve via the selective expansion of ecDNA-bearing cells. Through multi-regional sampling of tumors, we demonstrate that ecDNA contributes to the evolution of multifocality and increased intratumoral heterogeneity. EcDNA is present in 36% of UC tumors and correlates with an immunosuppressive phenotype and poor prognosis. Single-cell RNA sequencing analyses reveal that ecDNA+ malignant cells exhibit diminished expression of major histocompatibility complex 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.

Non-alcoholic fatty liver disease (NAFLD) is increasingly recognized for its global prevalence and potential progression to more severe liver diseases such as non-alcoholic steatohepatitis (NASH). The gut microbiota plays a pivotal role in the pathogenesis of NAFLD, yet the detailed characteristics and ecological alterations of gut microbial communities during the progression from non-alcoholic fatty liver (NAFL) to NASH remain poorly understood. Methods: In this study, we conducted a comparative analysis of gut microbiota composition in individuals with NAFL and NASH to elucidate differences and characteristics. We utilized 16S rRNA sequencing to compare the intestinal gut microbiota among a healthy control group (65 cases), NAFL group (64 cases), and NASH group (53 cases). Random forest machine learning and database validation methods were employed to analyze the data. Results: Our findings indicate a significant decrease in the diversity of intestinal flora during the progression of NAFLD (p < 0.05). At the phylum level, high abundances of Bacteroidetes and Fusobacteria were observed in both NAFL and NASH patients, whereas Firmicutes were less abundant. At the genus level, a significant decrease in Prevotella expression was seen in the NAFL group (AUC 0.738), whereas an increase in the combination of Megamonas and Fusobacterium was noted in the NASH group (AUC 0.769). Furthermore, KEGG pathway analysis highlighted significant disturbances in various types of glucose metabolism pathways in the NASH group compared to the NAFL group, as well as notably compromised flavonoid and flavonol biosynthesis functions. The study uncovers distinct microbiota characteristics and microecological changes within the gut during the transition from NAFL to NASH, providing insights that could facilitate the discovery of novel biomarkers and therapeutic targets for NAFLD.

Chromothripsis, a hallmark of cancer, is characterized by extensive and localized DNA rearrangements involving one or a few chromosomes. However, its genome-wide frequency and characteristics in urothelial carcinoma (UC) remain largely unknown. Here, by analyzing single-regional and multi-regional whole genome sequencing (WGS), we present the chromothripsis blueprint in 488 UC patients. Chromothripsis events exhibit significant intertumoral heterogeneity, being detected in 41% of UC patients, with an increase from 30% in non-muscle invasive disease (Ta/1) to 53% in muscle-invasive disease (T2-4). The presence of chromothripsis correlates with an unstable cancer genome and poor clinical outcomes. Analysis of multi-regional WGS data from 52 patients revealed pronounced intratumoral heterogeneity with chromothripsis events detectable only in specific tumor regions rather than uniformly across all areas. Chromothripsis events evolve under positive selection and contribute to tumor dissemination. This study presents a comprehensive genome-wide chromothripsis landscape in UC, highlighting the significance of chromothripsis in UC development.

Rationale: Extrachromosomal circular DNA is a hallmark of cancer, but its role in shaping the genome heterogeneity of urothelial bladder carcinoma (UBC) remains poorly understood. Here, we comprehensively analyzed the features of extrachromosomal circular DNA in 80 UBC patients. Methods: We performed whole-genome/exome sequencing (WGS/WES), Circle-Seq, single-molecule real-time (SMRT) long-read sequencing of circular DNA, and RNA sequencing (RNA-Seq) on 80 pairs of tumor and AT samples. We used our newly developed circular DNA analysis software, Circle-Map++ to detect small extrachromosomal circular DNA from Circle-Seq data. Results: We observed a high load and significant heterogeneity of extrachromosomal circular DNAs in UBC, including numerous single-locus and complex chimeric circular DNAs originating from different chromosomes. This includes highly chimeric circular DNAs carrying seven oncogenes and circles from nine chromosomes. We also found that large tumor-specific extrachromosomal circular DNAs could influence genome-wide gene expression, and are detectable in time-matched urinary sediments. Additionally, we found that the extrachromosomal circular DNA correlates with hypermutation, copy number variation, oncogene amplification, and clinical outcome. Conclusions: Overall, our study provides a comprehensive extrachromosomal circular DNA map of UBC, along with valuable data resources and bioinformatics tools for future cancer and extrachromosomal circular DNA research.

ABSTRACT Extrachromosomal circular DNAs (ecDNA) are focal gene amplifications frequently associated with cancer development and often indicating a poor prognosis. To understand the early dynamics of oncogene-carrying ecDNAs, we previously developed CRISPR-C, a tool for precise ecDNA generation by deleting specific chromosomal regions. Here, we adapted CRISPR-C to recreate tumor ecDNAs. This method also allowed us to enhance ecDNA generation efficiency by directly delivering Cas9 protein and sgRNAs as a ribonucleoprotein complex. By using the modified CRISPR-C, we successfully generated ecDNAs carrying oncogenes ( EGFR, CDK4, MDM2, MYC, MYCN, FGFR2, ABCB1, and DHFR ) in various human cell types. Furthermore, we demonstrated that our method could generate chimeric ecDNAs composed of target sequences from distant intra or inter-chromosomal regions. Using these generative ecDNA cell models, we studied the oncogene ecDNA expression and stability. The MDM2 expression was increased after CRISPR-C, while CDK4 was decreased indicating genomic-context dependent effect. The copy number of CRISPR-C generated CDK4 was ecDNA increased in cells after a long period of treatment with the CDK4 inhibitor palbociclib. Unlike CDK4, the CRISPR-C generated ABCB1 ecDNA was unstable in cells under normal growth conditions, but is stably retained when the cells were treated with colcemid, a recognized substrate for ABCB1. We thus provide valuable tools and an attractive platform for studying ecDNA biogenesisy and in vitro drug screening on ecDNA stability.

Primary endothelial cells (ECs), especially human umbilical vein endothelial cells (HUVECs), are broadly used in vascular biology. Gene editing of primary endothelial cells is known to be challenging, due to the low DNA transfection efficiency and the limited proliferation capacity of ECs. We report the establishment of a highly efficient and selection-free CRISPR gene editing approach for primary endothelial cells (HUVECs) with ribonucleoprotein (RNP) complex. We first optimized an efficient and cost-effective protocol for messenger RNA (mRNA) delivery into primary HUVECs by nucleofection. Nearly 100% transfection efficiency of HUVECs was achieved with EGFP mRNA. Using this optimized DNA-free approach, we tested RNP-mediated CRISPR gene editing of primary HUVECs with three different gRNAs targeting the HIF1A gene. We achieved highly efficient (98%) and biallelic HIF1A knockout in HUVECs without selection. The effects of HIF1A knockout on ECs' angiogenic characteristics and response to hypoxia were validated by functional assays. Our work provides a simple method for highly efficient gene editing of primary endothelial cells (HUVECs) in studies and manipulations of ECs functions.

Supplementary Figure Legends 1-4, Methods from Temporal Dissection of Tumorigenesis in Primary Cancers

Urine-based liquid biopsy has emerged as a non-invasive and effective tool for early screening and diagnosis of bladder cancer. This review provides a comprehensive overview of the current urine-based biomarkers and methods for the detection and monitoring of bladder cancer. We focus on biomarkers including tumour DNAs, proteins, microbiome, tumour RNAs, long non-coding RNAs, transfer RNA-derived fragments, messenger RNAs, microRNAs, circular RNAs, exosomes and extrachromosomal circular DNA. A systematic overview of technical considerations in developping urine biomarkers. Seven classes of urine biomarkers are summarised for bladder cancer detection and monitoring. Highlight of limitations and future improvements. image

Supplementary Figure 4 from Temporal Dissection of Tumorigenesis in Primary Cancers

Supplementary Figure 3 from Temporal Dissection of Tumorigenesis in Primary Cancers

Recent progress in CRISPR gene editing tools has substantially increased the opportunities for curing devastating genetic diseases. Here we compare in-frame deletion by CRISPR-based non-homologous blunt end joining (NHBEJ), homology-directed repair (HDR), and prime editing (PE, PE2, and PE3)-based correction of two Duchenne Muscular Dystrophy (DMD) loss-of-function mutations (c.5533G>T and c.7893delC). To enable accurate and rapid evaluation of editing efficiency, we generated a genomically integrated synthetic reporter system (VENUS) carrying the DMD mutations. The VENUS contains a modified enhanced green fluorescence protein (EGFP) gene, in which expression was restored upon the CRISPR-mediated correction of DMD loss-of-function mutations. We observed that the highest editing efficiency was achieved by NHBEJ (74–77%), followed by HDR (21–24%) and PE2 (1.5%) in HEK293T VENUS reporter cells. A similar HDR (23%) and PE2 (1.1%) correction efficiency is achieved in fibroblast VENUS cells. With PE3 (PE2 plus nicking gRNA), the c.7893delC correction efficiency was increased 3-fold. Furthermore, an approximately 31% correction efficiency of the endogenous DMD: c.7893delC is achieved in the FACS-enriched HDR-edited VENUS EGFP+ patient fibroblasts. We demonstrated that a highly efficient correction of DMD loss-of-function mutations in patient cells can be achieved by several means of CRISPR gene editing.

Supplementary Figure 2 from Temporal Dissection of Tumorigenesis in Primary Cancers

IntroductionThe gradual loss of motor neurons (MNs) in the brain and spinal cord is a hallmark of amyotrophic lateral sclerosis (ALS), but the mechanisms underlying neurodegeneration in ALS are still not fully understood.MethodsBased on 75 ALS-pathogenicity/susceptibility genes and large-scale single-cell transcriptomes of human/mouse brain/spinal cord/muscle tissues, we performed an expression enrichment analysis to identify cells involved in ALS pathogenesis. Subsequently, we created a strictness measure to estimate the dosage requirement of ALS-related genes in linked cell types.ResultsRemarkably, expression enrichment analysis showed that α- and γ-MNs, respectively, are associated with ALS-susceptibility genes and ALS-pathogenicity genes, revealing differences in biological processes between sporadic and familial ALS. In MNs, ALS-susceptibility genes exhibited high strictness, as well as the ALS-pathogenicity genes with known loss of function mechanism, indicating the main characteristic of ALS-susceptibility genes is dosage-sensitive and the loss of function mechanism of these genes may involve in sporadic ALS. In contrast, ALS-pathogenicity genes with gain of function mechanism exhibited low strictness. The significant difference of strictness between loss of function genes and gain of function genes provided a priori understanding for the pathogenesis of novel genes without an animal model. Besides MNs, we observed no statistical evidence for an association between muscle cells and ALS-related genes. This result may provide insight into the etiology that ALS is not within the domain of neuromuscular diseases. Moreover, we showed several cell types linked to other neurological diseases [i.e., spinocerebellar ataxia (SA), hereditary motor neuropathies (HMN)] and neuromuscular diseases [i.e. hereditary spastic paraplegia (SPG), spinal muscular atrophy (SMA)], including an association between Purkinje cells in brain and SA, an association between α-MNs in spinal cord and SA, an association between smooth muscle cells and SA, an association between oligodendrocyte and HMN, a suggestive association between γ-MNs and HMN, a suggestive association between mature skeletal muscle and HMN, an association between oligodendrocyte in brain and SPG, and no statistical evidence for an association between cell type and SMA.DiscussionThese cellular similarities and differences deepened our understanding of the heterogeneous cellular basis of ALS, SA, HMN, SPG, and SMA.

Supplementary Table 1 from Temporal Dissection of Tumorigenesis in Primary Cancers

Transposable elements (TEs) and transcription factors (TFs) are involved in the precise regulation of gene expression during the preimplantation stage. Activation of TEs is a key event for mammalian embryonic genome activation and preimplantation early embryonic development. TFs are involved in the regulation of drastic changes in gene expression patterns, but an inventory of the interplay between TEs and TFs during normal/abnormal human embryonic development is still lacking. Here we used single-cell RNA sequencing data generated from biparental and uniparental embryos to perform an integrative analysis of TE and TF expression. Our results showed that endogenous retroviruses (ERVs) are mainly expressed during the minor embryonic genome activation (EGA) process of early embryos, while Alu is gradually expressed in the middle and later stages. Some important ERVs (e.g., LTR5_Hs, MLT2A1) and Alu TEs are expressed at significantly lower levels in androgenic embryos. Integrative analysis revealed that the expression of the transcription factors CTCF and POU5F1 is correlated with the differential expression of ERV TEs. Comparative coexpression network analysis further showed distinct expression levels of important TFs (e.g., LEUTX and ZSCAN5A) in dizygotic embryos vs. parthenogenetic and androgenic embryos. This systematic investigation of TE and TF expression in human early embryonic development by single-cell RNA sequencing provides valuable insights into mammalian embryonic development.

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.

Additional file 7. Includes a table indicating which antibodies have been used in each figure, table of antibody information including ID, targeted gene, and antigen sequence, and a table of staining reagents.