Genome Biology最新文献

Background: Beta-thalassemia is among the most common monogenic disorders, posing a major global health challenge. Editing of genetic modifiers, such as BCL11A erythroid enhancer and HBG promoters, enhances fetal hemoglobin expression and confers major therapeutic potential. Double-strand-break (DSB)-independent genome editing tools, such as base editors (BE), are potentially safer and better suited for multiplexed application than DSB-dependent CRISPR/Cas technology. However, harmful on- and off-target events remain a concern and must be excluded before clinical application, including chromosomal rearrangements invisible to standard detection technologies.

Results: Using primary patient-derived CD34⁺ cells from three donors, we investigate simplex and duplex BE-based disruption of the BCL11A erythroid enhancer and the BCL11A binding site (-115 bp) on the HBG promoter for DNA-level and functional studies at the RNA, protein, and morphological level. Analyses include direct comparison to DSB-based editing, the current clinically applied standard, and CAST-seq to assess recombination events, allowing wider inferences on relative safety. RNA-seq analyses for clones of primary CD34⁺ cells across all treatments confirm peak HBG induction for duplex BE and comparable effects on apoptotic and immune response signatures. Overall, duplex BE produces robust γ-globin and fetal hemoglobin induction, improves functional correction over simplex editing and results in low incidence of genomic alterations in both target loci.

Conclusions: Duplex BE targeting both BCL11A erythroid enhancer and HBG promoter enables functional correction and genome integrity. Our study highlights the efficacy, safety, and therapeutic potential of the present duplex BE approach.

Alternative polyadenylation (APA) is a pervasive RNA-processing mechanism in eukaryotes that significantly promotes transcriptome and proteome diversity. Here we proposed PolyAseqTrap, an R package for probing polyA sites from diverse 3' sequencing data. PolyAseqTrap implements a polyA read prioritization strategy to determine precise positions of polyA sites. Particularly, it incorporates a transferrable cross-species deep learning model for mitigating the long-pending internal priming problem. Moreover, PolyAseqTrap employs a weighted density peak clustering method to reducing microheterogeneity impact in different species. We evaluated PolyAseqTrap using data from 16 different 3' sequencing techniques across multiple species, demonstrating the effectiveness and robustness of PolyAseqTrap.

Background: CRISPR/dCas9-based epigenome editing systems, including DNA methylation epimodifiers, have greatly advanced molecular functional studies, revolutionizing their precision and applicability. Despite their promise, challenges such as the magnitude and stability of the on-target editing and unwanted off-target effects underscore the need for improved tool characterization and design.

Results: We systematically compare specific targeting and genome-wide off-target effects of available and novel dCas9-based DNA methylation editing tools over time. We demonstrate that multimerization of the catalytic domain of DNA methyltransferase 3A enhances editing potency but also induces widespread, early methylation deposition at low-to-medium methylated promoter-related regions with specific gRNAs and also with non-targeting gRNAs. A small fraction of the methylation changes associated with transcriptional dysregulation and mapped predominantly to bivalent chromatin associating both with transcriptional repression and activation. Additionally, specific non-targeting control gRNAs cause pervasive and long-lasting methylation-independent transcriptional alterations particularly in genes linked to RNA and energy metabolism. CRISPRoff emerges as the most efficient tool for stable promoter targeting, with fewer and less stable off-target effects compared to other epimodifiers but with persistent transcriptome alterations.

Conclusions: Our findings highlight the delicate balance between potency and specificity of epigenome editing and provide critical insights into the design and application of future tools to improve their precision and minimize unintended consequences.

Background: Long dismissed as mere genomic parasites, transposable elements (TEs) are now recognized as major drivers of genome evolution. TEs serve as a source of cell-type specific cis-regulatory elements, influencing gene expression and observable phenotypes. However, the precise TE regulatory roles in different contexts remain largely unexplored and the impact of TEs on transcriptional regulatory networks and contribution to disease risk is likely deeply underestimated.

Results: Using a multimapper-aware strategy, we systematically characterize the epigenetic profile of TEs in human cell systems modeling neural development. This analysis reveals that MER57E3, a primate-specific TE subfamily, exhibits strong enrichment for active, and absence of repressive, histone modifications across six cultured human neural cell types. MER57E3 copies are predominantly located near zinc finger genes and enriched for homeodomain motifs recognized by brain-specific transcription factors, including GBX1 and BSX. Upon CRISPR interference (CRISPRi) targeting specific MER57E3 copies, RNA-seq analysis demonstrates downregulation of the key neurogenesis-related genes PAX6 and NEUROG2.

Conclusions: Our data indicate that members of the MER57E3 TE subfamily regulate the expression of critical neurogenesis genes during neural progenitor cell (NPC) development. Moreover, this study emphasizes the importance of investigating TEs, offering new insights into how their epigenetic dysregulation may contribute to pathogenesis of neurodevelopmental disorders.

Background: The ruminant gastrointestinal epithelium harbors a diverse and functionally critical remains poorly characterized microbial community due to persistent host-derived DNA contamination in metagenomic studies.

Results: We develop Dilute-MetaSeq (dilution-based metagenomic sequencing), a novel, metagenomic workflow integrating gradient dilution with multiple displacement amplification. Dilute-MetaSeq reduces host DNA interference by 52.4-fold and achieves > 90% microbial sequencing efficiency to assess gastrointestinal epithelium-associated microbiome. This enables the construction of the microbial genome atlas of gastrointestinal epithelium (MGA-GE). This comprehensive resource, comprising 1,907 nonredundant prokaryotic and 5,603 viral genomes, reveals extraordinary microbial diversity and novelty, with 41.4% of prokaryotic and 99.9% of viral genomes representing taxonomically unclassified lineages. Spatial profiling identifies the rumen and reticulum as a biodiversity hotspot dominated by epithelium-adapted Butyrivibrio and methylotrophic Methanomassiliicoccales, while functional annotation uncovers 1,200 biosynthetic gene clusters (primarily RiPPs and NRPSs) and 1,212 viral auxiliary metabolic genes linked to host metabolism modulation. Pangenome analysis of 987 strains, including a novel Butyrivibrio clade with reduced genome sizes, elevated GC content, and butyrate synthesis from amino acid-derived substrates (e.g., glutarate, lysine), highlights metabolic adaptations to the nutrient-scarce epithelial niche compared to digesta-associated microbes.

Conclusions: Collectively, the MGA-GE provides transformative insights into host-microbe-virus interactions and establishes a foundation for developing microbiome-based intervention strategies to enhance ruminant health, agricultural productivity, and bioactive discovery.

We introduce SCITO-seq2, an enhanced successor to SCITO-seq that integrates probe-based RNA detection with the established ultra-high-throughput protein profiling. SCITO-seq2 achieves robust quantification of transcripts and surface proteins across more than 100,000 cells, with a shared pool barcoding strategy ensuring precise matching of molecular profiles within multiplexed droplets. SCITO-seq2 is compatible with cell hashing technology, allowing efficient sample multiplexing. We demonstrate its utility in autoimmune diseases, including childhood systemic lupus erythematosus and CTLA4 haploinsufficiency with autoimmune infiltration, enabling the detection of minor immune clusters and disease-specific protein signatures. This platform establishes a scalable, streamlined, and cost-effective next-generation single-cell multi-omics workflow.

Nanopore sequencing enables real-time, long-read analysis by processing raw signals as they are produced. A key step, segmentation of signals into events, is typically handled algorithmically, struggling in noisy regions. We present Campolina, a first deep-learning framework for accurate segmentation of raw nanopore signals. Campolina uses a convolutional model to identify event boundaries and significantly outperforms the traditional Scrappie algorithm on R9.4.1 and R10.4.1 datasets. We introduce a comprehensive evaluation pipeline and show that Campolina aligns better with reference-guided ground-truth segmentation. We show that integrating Campolina segmentation into real-time frameworks, Sigmoni and RawHash2, improves their performance while maintaining time efficiency.

Background: IscB (Insertion sequences Cas9-like OrfB) represents a novel class of RNA-guided nucleases, approximately one-third the size of Cas9 proteins. Despite the limited natural efficiency in eukaryotic cells, recent advances have led to the engineering of several IscBs for mammalian genome editing.

Results: In this study, we screen and identify high-activity IscB variants for rice. A version of pIscB-v3, combining enOgeuIscB and ωRNA-v13, demonstrated superior mutagenesis efficiency compared to other systems. The average editing efficiency of pIscB-v3 is 17.61% from ten endogenous targets, and we obtain edited lines in up to 83.33% of T0 generation with 33.33% of homozygous and bi-allelic mutations. Further analysis reveals that pIscB-v3 exhibits high editing specificity and relaxed target-adjacent motif (TAM) compatibility in rice. Beyond gene knockout systems, we develop cytosine base editors (CBEs) and adenine base editors (ABEs) from pIscB-v3. We find that the ssDNA-targeting SCP1.201 family deaminase Sdd7 outperformed human APOBEC3A in IscB-CBEs for C-to-T conversions in rice. The Sdd7-nIscB achieves precise edits in 22.92% of lines on average, with a maximum frequency of 47.92%. Additionally, TadA8e-nIscB exhibits limited activity. However, fusing an extra copy of TadA-8e to either terminus of TadA8e-nIsc significantly enhances A-to-G conversions.

Conclusions: Collectively, our results demonstrate the robust capabilities of IscB to develop an efficient and versatile miniature plant genome editing toolkit to substantially facilitate crop breeding.

Background: Processing of archaeal 16S and 23S rRNAs is believed to involve excision of individual rRNAs from polycistronic precursors, circularization of excised rRNAs, and re-linearization before the incorporation into ribosomes. However, all the knowledge is derived from several isolated species, leaving open the possibility that different processes may occur in other archaeal groups.

Results: Here, we investigate rRNAs from diverse and mostly uncultivated archaea. Sequencing of total cellular RNA from eight phylum-level lineages indicates that archaeal circular 23S rRNA transcript abundances vastly exceed those of linear counterparts, and linear versions are often undetectable. As the majority of rRNAs derive from mature ribosomes, the data suggest that ribosomes contain circular 23S rRNAs. Thus, we directly sequence RNA extracted from isolated ribosomes of a model archaeon, Methanosarcina acetivorans, and confirm that the 23S rRNAs in the ribosomes are circular. Structural modeling places the 5' and 3' ends of the linear precursors of archaeal 23S rRNAs in close proximity to form a GNRA tetraloop (in which N is A, C, G, or U and R is A or G), consistent with their existence as circular molecules. We also confirm the existence of circular 16S rRNA intermediates in transcriptomes of most archaea, yet a circular form is not evident in some distinct archaeal groups, suggesting that certain archaea do not circularize 16S rRNA during processing.

Conclusions: Our findings uncover unexpected variations in the processing required to generate mature rRNAs and the conformation of functional molecules in archaeal ribosomes.