The transcription repressor BCL11A, which governs the switch from fetal to adult hemoglobin during development, is the target of the first FDA-approved CRISPR/Cas9 gene-editing therapy in humans. By targeting BCL11A, fetal hemoglobin expression is de-repressed to substitute for defective adult hemoglobin in inherited diseases including beta-thalassemia and sickle-cell anemia. BCL11A has six CCHH-type zinc-fingers of which domains 4-6 are necessary and sufficient for dsDNA binding. Here, we focus on the CCHC-type ZNF at the N-terminus of BCL11A (residues 46-72), Z0, thought to modulate oligomerization of the transcription repressor. Using NMR and CD spectroscopy, Z0 is shown to be a thermostable CCHC zinc-finger with a pM dissociation constant for zinc. The NMR structure of Z0 has a prototypical beta-beta-alpha fold, with a hydrophobic knob comprising about half the structure. The unusual proportion of hydrophobic residues in Z0 led us to investigate if this is more general in zinc-fingers that do not bind dsDNA. We used the ZF and WebLogo servers to examine sequences of zinc fingers with demonstrated DNA-binding function, non-binders, and the CCHC-type family of protein-binders. DNA-binders are distinguished by contiguous stretches of high-scoring zinc-fingers. Non-DNA-binders show a depletion of polar residues at the positions expected to contact nucleotides and increased divergence from sequence consensus making these domains more likely to be annotated as atypical, degenerate, or to be missed as zinc-fingers. We anticipate these sequence patterns will help distinguish DNA-binders from non-binders, an open problem in the functional understanding of zinc-finger motifs.

The study investigates the impact of targeted chromosome engineering on telomere dynamics, chromatin structure, gene expression, and phenotypic stability in Arabidopsis thaliana . Using precise CRISPR/Cas-based engineering, reciprocal translocations of chromosome arms were introduced between non-homologous chromosomes. The subsequent homozygous generations of plants were assessed for phenotype, transcriptomic changes and chromatin modifications near translocation breakpoints, and telomere length maintenance. Phenotypically, translocated lines were indistinguishable from wild-type plants, as confirmed through morphological assessments and principal component analysis. Gene expression profiling detected minimal differential expression, with affected genes dispersed across the genome, indicating negligible transcriptional impact. Similarly, ChIPseq analysis showed no substantial alterations in the enrichment of key histone marks (H3K27me3, H3K4me1, H3K56ac) near junction sites or across the genome. Finally, bulk and arm-specific telomere lengths remained stable across multiple generations, except for minor variations in one translocation line. These findings highlight the remarkable genomic and phenotypic robustness of A. thaliana despite large-scale chromosomal rearrangements. The study offers insights into the cis -acting mechanisms underlying chromosome arm-specific telomere length setting and establishes the feasibility of chromosome engineering for studies of plant genome evolution and crop improvement strategies. Significance statement This study demonstrates the robustness of Arabidopsis thaliana in maintaining telomere stability, chromatin integrity, and wild-type phenotype despite large-scale chromosomal translocations. It underscores the potential of chromosome engineering in advancing genome evolution research and crop improvement, contributes to a deeper understanding of genome dynamics, and opens new avenues for precision breeding in plants.

Background: and Objectives. Cartilage repair remains a critical challenge in orthopaedic medicine due to the tissue's limited self-healing ability, contributing to degenerative joint conditions such as osteoarthritis (OA). In response, regenerative medicine has developed advanced therapeutic strategies, including cell-based therapies, gene editing, and bioengineered scaffolds, to promote cartilage regeneration and restore joint function. This narrative review aims to explore the latest developments in cartilage repair techniques, focusing on mesenchymal stem cell (MSC) therapy, gene-based interventions, and biomaterial innovations. It also discusses the impact of patient-specific factors, such as age, defect size, and cost-efficiency, on treatment selection and outcomes. Methods. The review synthesises findings from recent clinical and preclinical studies published within the last five years, retrieved from PubMed, Scopus, and Web of Science databases. The search targeted key terms such as "cartilage repair," "stem cell therapy," "gene editing," "biomaterials," and "tissue engineering." Key Findings. Advances in MSC-based therapies, including autologous chondrocyte implantation (ACI) and platelet-rich plasma (PRP), have demonstrated promising regenerative potential. Gene-editing tools like CRISPR/Cas9 have facilitated targeted cellular modifications, while novel biomaterials such as hydrogels, biodegradable scaffolds, and 3D-printed constructs have improved mechanical support and tissue integration. Additionally, biophysical stimuli like low-intensity pulsed ultrasound (LIPUS) and electromagnetic fields (EMFs) have enhanced chondrogenic differentiation and matrix production. Treatment decisions are influenced by patient age, cartilage defect size, and financial considerations, highlighting the need for personalised and multimodal approaches. Conclusions. Combining regenerative techniques, including cell-based therapies, gene modifications, and advanced scaffolding, offers a promising pathway toward durable cartilage repair and joint preservation. Future research should focus on refining integrated therapeutic protocols, conducting long-term clinical evaluations, and embracing personalised treatment models driven by artificial intelligence and predictive algorithms.

Regulatory T cells (Tregs) play a central role in immune regulation and tolerance. The transcription factor FOXP3 is a master regulator of Tregs in both humans and mice. Mutations in FOXP3 lead to the development of IPEX syndrome in humans and the scurfy phenotype in mice, both of which are characterized by fatal systemic autoimmunity. Additionally, Treg dysfunction and FOXP3 expression instability have been implicated in non-genetic autoimmune diseases, including graft-versus-host disease, inflammatory bowel disease, rheumatoid arthritis, and multiple sclerosis. Recent investigations have explored FOXP3 expression in allergic diseases, revealing Treg alterations in food allergies, asthma, and atopic dermatitis. This review examines the multifaceted roles of FOXP3 and Tregs in health and various pathological states including autoimmune disorders, allergic diseases, and cancer. Additionally, this review focuses on the impact of recent technological advancements in facilitating Treg-mediated cell and gene therapy approaches, including CRISPR/Cas9-based gene editing. The critical function of FOXP3 in maintaining immune homeostasis and tolerance to both self-antigens and alloantigens has been emphasized. Considering the potential involvement of Tregs in allergic diseases, pharmacological interventions and cell-based immunomodulatory strategies may offer promising avenues for developing novel therapeutic approaches in this field.

Environmental biosecurity challenges are worsening for aquatic ecosystems as climate change and increased anthropogenic pressures facilitate the spread of invasive species, thereby broadly impacting ecosystem composition, functioning, and services. Environmental DNA (eDNA) has transformed traditional biomonitoring through detection of trace DNA fragments left by organisms in their surroundings, primarily by application of the quantitative polymerase chain reaction (qPCR). However, qPCR presents challenges, including limited portability, reliance on precise thermal cycling, and susceptibility to inhibitors. To address these challenges and enable field-deployable monitoring, isothermal amplification techniques such as Recombinase Polymerase Amplification (RPA) paired with Clustered Regularly Interspaced Short Palindromic Repeats and associated proteins (CRISPR-Cas) have been proposed as alternatives. We report here the development of CORSAIR ( C RISPR-based envir O nmental biosu R veillance a S sisted via A rtificial I ntelligence guide- R NAs), that harnesses the programmability of the CRISPR-Cas technology, RPA and the artificial intelligence (AI)-based tool Activity-informed Design with All-inclusive Patrolling of Targets (ADAPT) to deploy a swift RPA-CRISPR-Cas13a-based method that detects eDNA from two invasive species as proof of concept: Sabella spallanzanii and Undaria pinnatifida . CORSAIR showcased a robust, streamlined method augmented by ADAPT, reaching a high specificity when tested against co-occurring species and a 100% agreement with 12 PCR-benchmarked eDNA samples, reaching a sensitivity of 0.34 copies uL -1 in 1 hour with a cost of 3.5 USD per sample; thus highlighting CORSAIR as a powerful environmental biosurveillance platform for environmental nucleic acid detection. Graphical abstract

RNA velocity has emerged as a popular approach for modeling cellular change along the phenotypic landscape but routinely omits regulatory interactions between genes. Conversely, methods that infer gene regulatory networks (GRNs) do not consider the dynamically changing nature of biological systems. To integrate these two currently disconnected fields, we present RegVelo, an end-to-end dynamic, interpretable, and actionable deep learning model that learns a joint model of splicing kinetics and gene regulatory relationships and allows us to perform in silico perturbation predictions. When applied to datasets of the cell cycle, human hematopoiesis, and murine pancreatic endocrinogenesis, RegVelo demonstrates superior predictive power for interactions and perturbation simulations, for example, compared to methods that focus solely on dynamics or GRN inference. To leverage RegVelo’s full potential, we studied the dynamics of zebrafish neural crest development and underlying regulatory mechanisms using our Smart-seq3 dataset and shared gene expression and chromatin accessibility measurements. Using RegVelo’s in silico perturbation predictions, validated by CRISPR/Cas9-mediated knockout and single-cell Perturb-seq, we establish transcription factor tfec as an early driver and elf1 as a novel regulator of pigment cell fate and propose a gene-regulatory circuit involving tfec and elf1 interactions via the toggle-switch model.

Abstract Knocking out the oncogene UBC12 with Crispr Cas9 can inhibit the Neddylation pathway and lead to excellent anticancer effect. However, how to deliver Crispr Cas9 into tumor cells is an urgent scientific problem. Based on the application of polyethyleneimine and its derivative (GMP in vivo-jetPEI) in clinical trials, we constructed fluorine-containing PEI to deliver Crispr Cas9 plasmid. In order to reduce the off-target effect of Crispr Cas9 nanomedicine, the targeting molecule TLS11a aptamer was modified on the surface of genetic nanomedicine to improve the efficiency of initiative. The experimental results showed that the released plasmid of Crispr Cas9 inhibited Neddylation by knocking out UBC12, thus achieving the goal of inhibiting the growth of tumor cells. The design of this genetic nanomedicine provides a scientific basis for the construction of non-viral genetic nanomedicine.

Abstract Cancer hallmarks describe key physiological characteristics that distinguish cancers from normal tissues. The temporal order in which these hallmarks appear during cancer pathogenesis is of interest from both evolutionary and clinical perspectives but has not been investigated before. Here, we order hallmarks based on the allele frequency and selective advantage of mutations in cancer hallmark genes across > 10K untreated primary tumors and > 8K healthy tissues. Using this novel approach, we identified a common evolutionary trajectory for 27 of 32 cancer types with genomic instability appearing first and immune evasion appearing last. We demonstrated widespread positive selection in cancer and strong negative selection in normal tissues for all hallmarks. Notable exceptions to the hallmark ordering in tumors were melanomas (uveal and skin) suggesting that strong environmental factors could disrupt common evolutionary paths. Clustering of hallmark trajectories across patients revealed 2 clusters defined by early or late genomic instability, with differential prognosis. We finally validated our results in about 3K primary tumors from the PCAWG consortium. Our study is the first to identify the temporal order of cancer hallmarks during tumorigenesis and demonstrate a prognostic value that could be exploited for early detection and risk stratification across multiple cancer types.

Precise control of gene expression is essential for cellular function, but the mechanisms by which enhancers communicate with promoters to coordinate this process are not fully understood. While sequence-based deep learning models show promise in predicting enhancer-driven gene expression, experimental validation and human-interpretable mechanistic insights lag behind. Here, we present EXTRA-seq , a novel EXT ended R eporter A ssay followed by seq uencing designed to quantify enhancer activity in endogenous contexts over kilobase-scale distances. We demonstrate that EXTRA-seq can be targeted to disease-relevant loci and captures expression changes at the resolution of individual transcription factor binding sites, enabling mechanistic discovery. Using engineered synthetic enhancer-promoter combinations, we reveal that the TATA-box acts as a dynamic range amplifier, modulating expression levels in function of enhancer strength. Importantly, we find that integrating state-of-the-art deep learning models with plasmid-based enhancer assays improves the prediction of gene expression as measured by EXTRA-seq. These findings open new avenues for predictive modeling and therapeutic applications. Overall, our work provides a powerful experimental platform to interrogate the complex interplay between enhancers and promoters, bridging the gap between in silico predictions and human-interpretable biological mechanisms.

The CRISPR/Cas9 system has become a powerful tool for molecular design breeding in livestock such as sheep. However, the efficiency of the Cas9 system combined with zygote microinjection remains suboptimal. In this study, mature sheep oocytes were used for microinjection to assess the impact of various factors on Cas9 editing efficiency. We found that the in vitro maturation efficiency of oocytes is influenced by environmental factors such as air temperature, pressure, and humidity. Our results indicate that high-efficiency gene editing can be achieved when targeting the SOCS2, DYA, and TBXT genes, using a microinjection mixture with a concentration of 10 ng/μl Cas9 and sgRNA. By optimizing the injection capillary, we significantly reduced the oocyte invalidation rate post-microinjection to 3.1–5.3%. Furthermore, we observed that using either Cas9 protein or mRNA in the microinjection process resulted in different genotypes in the edited oocytes. Importantly, parthenogenetic activation did not appear to affect the editing efficiency. Using this high-efficiency system, we successfully generated SOCS2 or DYA gene-edited sheep, with all lambs confirmed to be genetically modified. This study presents a highly efficient method for producing gene-edited sheep, potentially enabling more precise and effective strategies for livestock breeding.

ABSTRACT Successful transgenesis in model organisms has dramatically helped us understand gene function, regulation, genetic networks, and potential applications. Here, we introduce the universal single-copy knock-in system (Universal SKI System or U-SKI), designed for inserting any transgene by CRISPR/Cas9 in the Caenorhabditis elegans genome. The Universal SKI System takes advantage of a plasmid (pSKI), which can also be used for extrachromosomal arrays, to facilitate the insertion of a transgene at specific safe harbor loci on each autosomal chromosome. The pSKI plasmid contains multiple restriction sites for easy cloning and serves as a CRISPR/Cas9-based insertion repair template because it has two synthetic and long homology arms that recombine with the SKI cassettes. This system also uses a single crRNA guide, which acts as a Co-CRISPR enrichment marker. Overall, the Universal SKI System is highly flexible; with the same Universal SKI cassette on each autosome, researchers can select the insertion site and streamline tracking while reducing the complexity of expressing single-copy transgenes in C. elegans .

Background De novo shoot regeneration involves reconstitution of stem cells under control of stem cell regulators. The WUSCHEL (WUS) transcription factor acts as a master regulator of de novo shoot regeneration controlling genotype-dependent regeneration efficiency. Previously we found that potato plants overexpressing the StCLE4 gene encoding Solanum tuberosum CLAVATA3/EMBRYO SURROUNDING REGION-RELATED peptide hormone exhibited a wus -like phenotype with inhibited growth of the shoot apical meristem. We hypothesized that StCLE4 could also modulate de novo shoot regeneration. Methods and results In this study the role of the StCLE4 peptide in de novo shoot regeneration was studied by CRISPR/Cas9 mutant analysis, evaluation of overexpression effects and gene expression analysis. We found that cle4 mutation enhanced shoot regeneration in potato. Conclusions We have found a gene in potato, the loss of which can lead to increased shoot formation. These results can be used to improve the efficiency of shoot regeneration in potato.

Summary Chitin triggers localised and systemic plant immune responses, making it a promising treatment for sustainable disease resistance. However, the precise molecular mechanisms underlying chitin-induced systemic effects in plants remain unknown. In this study, we investigated the effects of soil amendment with crab chitin flakes (hereafter chitin) on pattern-triggered immunity (PTI) and systemic disease resistance in various plant species. We found that soil amendment with chitin potentiates PTI and disease resistance against the bacterial pathogen Pseudomonas syringae pv. tomato DC3000 in lettuce, tomato, and Arabidopsis as well as against the fungal pathogen Blumeria graminis causing powdery mildew in wheat. Using micrografting in Arabidopsis, we demonstrated that this systemic effect is dependent on active chitin perception in the roots. We also showed that induced systemic resistance (ISR) and pattern-recognition receptors (PRRs)/co-receptors, but not systemic acquired resistance (SAR), are involved in the systemic effects triggered by chitin soil amendment. This systemic effect correlated with the transcriptional up-regulation of key PTI components in distal leaves upon chitin soil amendment. Notably, chitin-triggered systemic immunity was independent of microbes present in soil or chitin flakes. Together, these findings contribute to a better understanding of chitin-triggered systemic immunity, from active chitin perception in roots to the potentiation of PTI in the leaves, ultimately priming plants to mount enhanced defense responses against pathogen attacks. Our study provides valuable insights into the molecular mechanisms of chitin soil amendment and resulting induced immunity, and highlights its potential use for sustainable crop protection strategies.

Autophagy sustains cellular health by recycling damaged or excess components through autophagosomes. It is mediated by conserved ATG proteins, which coordinate autophagosome biogenesis and selective cargo degradation. Among these, the ubiquitin-like ATG8 protein plays a central role by linking cargo to the growing autophagosomes through interacting with selective autophagy receptors. Unlike most ATG proteins, the ATG8 gene family is significantly expanded in vascular plants, but its functional specialization remains poorly understood. Using transcriptional and translational reporters in Arabidopsis thaliana , we revealed that ATG8 isoforms are differentially expressed across tissues and form distinct autophagosomes within the same cell. To explore ATG8 specialization, we generated the nonuple Δatg8 mutant lacking all nine ATG8 isoforms. The mutant displayed hypersensitivity to carbon and nitrogen starvation, coupled with defects in bulk and selective autophagy as shown by biochemical and ultrastructural analyses. Complementation experiments demonstrated that ATG8A could rescue both carbon and nitrogen starvation phenotypes, whereas ATG8H could only complement carbon starvation. Proximity labeling proteomics further identified isoform-specific interactors under nitrogen starvation, underscoring their functional divergence. These findings provide genetic evidence for functional specialization of ATG8 isoforms in plants and lay the foundation for investigating their roles in diverse cell types and stress conditions.

Honeybees, the world’s most important crop pollinators, are increasingly facing pollen starvation arising from agricultural intensification and climate change. Frequent flowering dearth periods and high-density rearing conditions weaken colonies, often leading to their demise. Beekeepers provide colonies with pollen substitutes, but these feeds cannot sustain brood production because they lack essential sterols found in pollen. Here, we describe a technological breakthrough in honeybee nutrition with wide-reaching impacts on global food security. We first measured the quantity and proportion of sterols found in honeybee tissues. Using this information, we genetically engineered a strain of the oleaginous yeast, Yarrowia lipolytica , to produce a mixture of essential sterols for bees and incorporated it into an otherwise nutritionally complete diet. Colonies fed exclusively with this diet reared brood for significantly longer than those fed diets without suitable sterols. Incorporating sterol supplements into pollen substitutes using this method will enable beekeepers to rear healthier, longer-lived colonies to meet the growing demands for global crop pollination. It could also reduce competition between bee species for access to natural floral resources, stemming the decline of wild bee populations.

Abstract Background Streptococcus thermophilus is invaluable in both of dairy factory and scientific research, and different S. thermophilus strains have different advantages in industry. As a result, fast, easy, biosafe, and widely applicable genome manipulation methods for S. thermophilus are of high demands. The traditional temperature-sensitive plasmid-based homologous recombination method is neither fast nor easy, the current natural transformation method is not widely applicable, and the recently established endogenous CRISPR-Cas system-assisted method is not easy in plasmid construction. Results In this project, we characterized the features of the natural transformation of S. thermophilus B-6 strain, and for the first time found that the DNA transport pathway activated at natural competent stage might be bidirectional. We optimized the natural transformation protocol to upgrade the transformation ratio of S. thermophilus B-6 from ~ 10 − 5 to ~ 10 − 2 . With the improved natural transformation procedure, we developed a fast, easy, biosafe, and plasmid-independent method for the genome manipulation of this strain. We also established a novel native CRISPR-Cas system-assisted genome manipulation pathway with a higher efficiency, which did not require any new plasmid construction. By the novel genome manipulation methods, we created different CRISPR-Cas system mutant strains and a recA overexpressing strain. Conclusions Our finding contributes to better understanding the features of natural competence. Our convenient and biosafe genome manipulation methods will be valuable for most of the S. thermophilus strains, and will contribute to the germplasm improvement of dairy industry. Besides, the S. thermophilus mutants we generated in this project will be useful in the future cellular metabolism investigations.

SUMMARY Extensive intratumor heterogeneity in glioblastoma (GBM) impedes successful treatment and complicates drug discovery as it is not obvious which cells a tumor is most dependent on. Here, we posit that single-cell-resolution transcriptomic data can be integrated with loss-of-function screens to identify the most critical cells to target within a tumor. We parsed CRISPR screen data from the Dependency Map (DepMap) Consortium and identified a GBM Dependency Signature (GDS) − 168 genes that are essential for GBM cell viability in vitro . Through similarity scoring of GDS transcriptomic profiles in single-cell RNA-sequencing (scRNA-seq) data and iterative hierarchical clustering, we identify and report here 3 single-cell vulnerability states (VS) characterized in 49 GBM tumors using both scRNA-seq and spatial transcriptomic data. These VS reflect single-cell gene dependencies and differ significantly in enrichment profiles and spatial distributions. Importantly, the proportion of VS in each GBM tumor is variable, suggesting a means of stratifying patients in clinical trials. Collectively, we have developed a novel computational pipeline to identify unique vulnerability states in GBM and other cancers, which can be used to identify existing or novel drugs for incurable diseases.

Resistance to BRAF and MAPK inhibitors is a significant challenge in melanoma treatment, driven by adaptive and acquired mechanisms that allow tumour cells to evade therapy. Here, we examined early signalling responses to single and combined BRAF and MAPK inhibition in a BRAFV600E, drug-sensitive melanoma cell line and a drug-resistant ARID1A-knockout (KO) derivative. ARID1A, frequently mutated in melanoma, is associated with resistance and immune evasion. Using an innovative systems biology approach that integrates transcriptomics, proteomics, phosphoproteomics, and functional kinomics through matrix factorization and network analysis, we identified key signalling alterations and resistance mechanisms. We found that ARID1A-KO cells exhibited transcriptional rewiring, sustaining MAPK1/3 and JNK activity post-treatment, bypassing feedback sensitivity observed in parental cells. This rewiring suppressed PRKD1 activation, increased JUN activity—a central resistance network node—and disrupted PKC dynamics through elevated basal RTKs (e.g., EGFR, ROS1) and Ephrin receptor activity post-treatment. ARID1A mutations also reduced HLA-related protein expression and enriched extracellular matrix components, potentially limiting immune infiltration and reducing immunotherapy efficacy. Our graph-theoretical multi-omics approach uncovered novel resistance-associated signalling pathways, identifying PRKD1, JUN, and NCK1 as critical nodes. While receptor activation redundancies complicate single-target therapies, they also present opportunities for combination strategies. This study highlights ARID1A’s role in reshaping signalling and immune interactions, offering new insights into melanoma resistance mechanisms. By identifying actionable targets, including JUN and immune pathways, we provide a foundation for developing integrated therapeutic strategies to overcome resistance in BRAF/MAPK inhibitor-treated melanoma. One sentence summary This study reveals how ARID1A-mediated transcriptional rewiring drives resistance to MAPK inhibitors in melanoma by altering signalling pathways, immune interactions, and receptor dynamics, highlighting potential targets for combinatorial therapies.

The genus Streptomyces are valuable producers of antibiotics and other pharmaceutically important bioactive compounds. Advances in molecular engineering tools, such as CRISPR, has provided some access to the metabolic potential of Streptomyces , but efficient genetic engineering of strains is hindered by laborious and slow manual transformation protocols. In this paper, we present a semi-automated medium-throughput workflow for the introduction of recombinant DNA into Streptomyces spp. using the affordable and open-sourced Opentrons (OT-2) robotics platform. To increase the accessibility of the workflow we provide an open-source protocol-creator, ActinoMation. ActinoMation is a literate programming environment using Python in Jupyter Notebook. We validated the method by transforming Streptomyces coelicolor (M1152 and M1146), S. albidoflavus (J1047), and S. venezuelae (DSM40230) with the plasmids pSETGUS and pIJ12551. We demonstrate conjugation efficiencies of 3.33*10 −3 for M1152 with pSETGUS and pIJ12551; 2.96*10 −3 for M1146 with pSETGUS and pIJ12551; 1.21*10 −5 for J1047 with pSETGUS and 4.70*10 −4 with pIJ12551, and 4.97*10 −2 for DSM40230 with pSETGUS and 6.13*10 −2 with pIJ12551 with a false positive rate between 8.33% and 54.54%. Automation of the conjugation workflow improves consistency when handling large sample sizes that facilitates easy reproducibility on a larger scale.

The XPG endonuclease plays a crucial role in nucleotide excision repair (NER) and other genome maintenance pathways. Precise regulation of XPG recruitment and activity during DNA repair is essential to avoid erroneous DNA incisions and genomic instability. In this study, we employed live-cell imaging to investigate how XPG function is regulated during NER, focusing on its dynamic interactions with key factors involved in the pre- and post-incision steps. We found that TFIIH and XPA facilitate the recruitment and association of XPG with DNA damage, and that XPG localizes separately from TFIIH to UV-induced lesions. Furthermore, our results show that XPG’s dissociation from DNA damage is triggered by its own incision activity as well as by that of XPF. Additionally, the exonuclease EXO1 promotes XPG dissociation, likely by processing incised DNA, even in the absence of XPG-mediated incision. Our findings help to better understand the regulatory mechanisms that control XPG activity during NER and provide important insights into the complex dynamics of the repair process.

Several RNA viruses induce widespread degradation of cellular mRNAs upon infection; however, the biological significance and mechanistic details of this phenomenon remain unknown. Here, we make use of a model alphavirus, Sindbis virus (SINV), to fill this knowledge gap. We found that SINV triggers cellular RNA decay through the exonuclease XRN1 and the 5’-to-3’ degradation machinery (5-3DM). These proteins accumulate at viral replication organelles (VROs) and interact with the non-structural protein 1 (nsP1), bringing mRNA degradation into proximity with vRNA synthesis. Our data suggest that monophosphate nucleotides released by cellular RNA decay are recycled through the salvage pathway to feed viral replications. Our work thus reveals a fundamental connection between cellular mRNA degradation and viral replication via nucleotides repurposing. Research highlights 5’-3’ RNA decay is essential for the replication of a wide range of viruses. XRN1 directly interacts with transcripts which are degraded during infection. RNA decay factors and salvage pathway members localise to viral factories. Supplying nucleosides to several 5-3DM deficient cells facilitates SINV infection.

Precision breeding tools such as CRISPR-Cas genome editing can speed up innovation in plant biotechnology and boost crop yields. The challenge remains to efficiently apply precision breeding methods to plant gene regulation. Endogenous gene regulatory sequences are subject to complex transcriptional control, a bottleneck in altering gene expression patterns in a predictable way. Here we present the CRE.AI.TIVE platform, enabling upregulation of plant gene activity without a priori knowledge of individual cis-regulatory elements or their specific location. A predictive machine learning model underpinning the platform has been trained on a wide range of tissue specific transcriptomic and epigenomic coverage datasets from DNA sequence of 12 plant species, showing competitive performance on RNA-seq coverage prediction across all species. Our platform further combines in silico DNA sequence mutagenesis and a protoplast-based massively parallel reporter assay (MPRA). We demonstrate the platform’s functionality by mutagenesis of a proximal promoter of the tomato gene SlbHLH96 which yields predictions of variant gene activity in silico . 2,000 sequence candidates with varying predicted gene expression strength were validated with MPRA in plant protoplasts, identifying variants with significantly upregulated gene activity. A portion of functional sequence variants were further individually evaluated with a fluorescence reporter assay and were observed to contain a new order of known cis-regulatory elements. The CRE.AI.TIVE platform offers a first-of-its-kind scalable method of gene upregulation in plants with native DNA sequences without the need for CRE cataloguing and rational promoter design.

Single-cell CRISPR (Perturb-seq) screens have primarily relied on Cas9 whereas Cas12a, despite its unique effectiveness for multiplex guide expression, remains underexplored. This may be due to Cas12a’s guide RNA array (pre-crRNA) self-processing activity and the subsequent challenges associated with pre-crRNA sequence recovery. By developing modified pre-crRNA expression vectors and a degron-based Cas12a system, we overcome the self-processing constraint, allowing for accurate detection of pre-crRNAs at the single-cell level, thus greatly expanding possibilities for future Perturb-seq efforts.

Understanding cellular responses to genetic perturbations is essential for understanding gene regulation and phenotype formation. While high-throughput single-cell RNA-sequencing has facilitated detailed profiling of heterogeneous transcriptional responses to perturbations at the single-cell level, there remains a pressing need for computational models that can decode the mechanisms driving these responses and accurately predict outcomes to prioritize target genes for experimental design. Here, we present scLAMBDA, a deep generative learning framework designed to model and predict single-cell transcriptional responses to genetic perturbations, including single-gene and combinatorial multi-gene perturbations. By leveraging gene embeddings derived from large language models, scLAMBDA effectively integrates prior biological knowledge and disentangles basal cell states from perturbation-specific salient representations. Through comprehensive evaluations on multiple single-cell CRISPR Perturb-seq datasets, scLAMBDA consistently outperformed state-of-the-art methods in predicting perturbation outcomes, achieving higher prediction accuracy. Notably, scLAMBDA demonstrated robust generalization to unseen target genes and perturbations, and its predictions captured both average expression changes and the heterogeneity of single-cell responses. Furthermore, its predictions enable diverse downstream analyses, including the identification of differentially expressed genes and the exploration of genetic interactions, demonstrating its utility and versatility.

ABSTRACT More than 50 repeat expansion disorders have been identified, with long-read sequencing marking a new milestone in the diagnosis of these disorders. Despite these major achievements, the comprehensive characterization of short tandem repeats in a pathological context remains challenging, primarily due to their inherent characteristics such as motif complexity, high GC content, and variable length. In this study, our aim was to thoroughly characterize repeat expansions in two neuromuscular diseases: myotonic dystrophy type 1 (DM1) and oculopharyngodistal myopathy (OPDM) using CRISPR/Cas9-targeted long-read sequencing (Oxford Nanopore Technologies, ONT). We conducted precise analyses of the DM1 and OPDM loci, determining repeat size, repeat length distribution, expansion architecture and DNA methylation, using three different basecallers (Guppy, Bonito and Dorado). We demonstrated the importance of the basecalling strategy in repeat expansion characterization. We proposed guidelines to perform CRISPR-Cas9 targeted long-read sequencing (no longer supported by ONT), from library preparation to bioinformatical analyses. Finally, we showed, for the first time, somatic mosaicism, hypermethylation of LRP12 loci in symptomatic patients and changes in the repeat tract structure of OPDM patients. We propose a strategy based on CRISPR/Cas9-enrichment long-read sequencing for repeat expansion diseases, which could be readily applicable in research but also in diagnostic settings.