The regenerative response of retinal cells to injury and aging depends on the epigenomic plasticity that enables the dedifferentiation and neuronal differentiation capacities of Müller glial cells (MG). In mammals, this regenerative ability is extremely limited, and disruptions in epigenetic mechanisms, particularly those involving DNA methylation and demethylation, may underlie this restricted potential. To explore this possibility, we aimed to develop DNA methylation-targeting molecular tools to enhance the dedifferentiation and neurogenic capacity of primary MG cultures derived from mouse retina. Using CRISPR/dCas9-based gene regulation technology, we selectively and transiently inhibited Dnmt3a , a de novo DNA methyltransferase previously implicated in maintaining transcriptional repression. Our results show that Dnmt3a knockdown leads to sustained upregulation of pluripotency-associated genes, including Ascl1 , Lin28 , and Nestin , as measured by RT-qPCR and immunofluorescence. This epigenetic modulation also promoted increased cell proliferation and migration, both hallmarks of a regenerative response. Furthermore, Dnmt3a knockdown, either alone or in combination with neurogenic stimuli, induced MG to acquire neuronal-like morphologies and express the early neuronal marker βIII-tubulin. These findings suggest that Dnmt3a acts as a repressive regulator of MG plasticity, likely serving as an epigenetic barrier that counteracts injury-induced demethylation events. Overall, our study identifies Dnmt3a as a critical modulator of MG fate and highlights the potential of its targeted downregulation to facilitate reprogramming. By prolonging the transient progenitor-like state of MG, DNMT3a inhibition may serve as a complementary approach to unlock the neurogenic and regenerative potential of the mammalian retina, offering promising avenues for future therapeutic strategies. Author Summary Retinal damage caused by injury, disease, or aging can lead to vision loss and ultimately blindness. Unlike mammals, the small freshwater zebrafish possesses a remarkable ability to regenerate its retina and restore vision after injury. Extensive research has focused on uncovering the molecular mechanisms behind this regenerative process in zebrafish, with the goal of understanding what is absent or suppressed in the mammalian retina. It is now well established that this regenerative capacity depends on a specific type of retinal cell: Müller glia. These cells can undergo dedifferentiation, a process in which they lose their specialized function, morphology, and gene expression profile. This is followed by neuronal differentiation, allowing them to replace lost neurons with newly generated ones. In recent years, numerous molecules and molecular pathways have been identified that may limit regenerative potential in mammals. In this study, we developed a molecular tool to specifically block one of these inhibitory factors, DNMT3a, a DNA methyltransferase involved in epigenetic repression. We demonstrate that in the absence of DNMT3a, mouse Müller glia can more efficiently dedifferentiate and subsequently adopt neuronal-like characteristics. These findings suggest that DNMT3a acts as a barrier to retinal regeneration and may represent a promising target for future therapeutic strategies aimed at promoting neural repair in the mammalian retina.

We know little about the fitness landscapes of bacterial operators, regulatory DNA elements that are crucial to regulate metabolic genes like those of the lac operon for lactose utilization. For example, we do not know whether adaptive evolution could easily create strong operators from weak ones or from non-regulatory DNA. To find out, we used CRISPR-Cas-assisted genome editing, bulk competition, and high-throughput sequencing to map the fitness landscape of more than 140,000 lac operator variants in two chemical environments that harbor lactose or glycerol as sole carbon sources. Both landscapes are highly rugged and contain thousands of fitness peaks, which allow only 2 percent of evolving populations to reach a high fitness peak. The landscapes share only 15 percent of fitness peaks. Our work illustrates that landscape ruggedness caused by epistasis can represent an important obstacle to adaptive evolution of regulatory sequences. It also shows that a simple environmental change can substantially affect fitness landscape topography.

Recent advances in prime editing technologies using CRISPR modules fused with reverse transcriptase (RT) have enabled efficient and precise reprogramming of target genomic sequences. Twin prime editing using two coordinated prime editor complexes is a promising strategy for inducing extensive genomic modifications via reverse transcribed complementary templates. However, current twin prime editing systems still require improvements in editing efficiency, accuracy, and intended edit predictability. Here, efficiency and precision of twin prime editing were enhanced via engineering and optimizing conventional SpCas9(H840A)-RT-based prime editor components. A La domain–fused prime editor (La-SpCas9(H840A)-RT) and optimized pegRNAs were developed, achieving a 1.75 ± 0.21-fold increase in gene editing efficiency at multiple genomic loci in human-derived cell lines without increasing off-target activity. La-SpCas9(H840A)-RT facilitated efficient ∼2.8 kb GFP transgene knock-in at target loci and eliminated the expanded polyQ tract in ATXN3 in patient-derived mutant cell lines modeling spinocerebellar ataxia type 3. The advanced twin prime editing platform expands genome engineering capabilities beyond existing CRISPR-based systems and holds great promise for diverse biotechnological and therapeutic applications.

Targeted delivery of macromolecular therapeutics holds great promise for overcoming the limitations of conventional small molecules, enabling modulation of protein-protein interactions and precise genome editing. However, efficient, safe, and cell type-specific delivery remains a major challenge. To address this, we developed a modular platform for synthesizing heterotrifunctional bio-orthogonal macromolecular conjugates (BMCs) by engineering diverse combinations of targeting ligands, cell-penetrating peptides (CPPs), and bioactive cargos. We optimized facile bioconjugation chemistries to generate BMCs with improved yields, structural integrity, and activity. Modular BMCs accommodate diverse components, including antibodies and receptor ligands for targeting, CPPs for intracellular trafficking, and optical probes, therapeutic peptidomimetics, and CRISPR-Cas9 nuclease as cargos to confer specific biological activities. We assayed their utility across multiple applications: BMCs with fluorescently labeled cargo revealed endosomal escape and intracellular accumulation; peptidomimetic MYB transcription factor inhibitor BMCs exhibited potent anti-leukemic activity against acute myeloid leukemia cells; and Cas9 BMCs achieved rapid delivery and cell type-specific gene editing in human cells. The BMC approach enables customizable delivery of functional macromolecules, nominating BMCs as a broadly applicable platform for biomedical applications. One-Sentence Summary The establishment of modular platform for synthesizing bio-orthogonal macromolecular conjugates (BMC) enabled fast and targeted delivery of membrane-impermeant macromolecular drugs.

Plant cysteine oxidases (PCOs) are O 2 -sensing enzymes that play an important role in plant responses to low oxygen (hypoxia). PCO-catalysed dioxygenation of the N-terminal Cys of substrates, including Group VII Ethylene Response Factors (ERVIIs), targets them for degradation via the Cys/Arg N-degron pathway, however these substrates are stabilized in hypoxia due to reduced PCO activity. When plants are flooded, submergence-induced hypoxia results in ERFVII-mediated upregulation of hypoxia responsive genes that reconfigure plant metabolism and allow short-term resilience to the conditions. However, the increasing frequency and duration of flood events requires strategies to improve plant flood resilience, particularly amongst agronomic crops. One possibility is to prolong the stability of ERFVIIs by engineering the PCOs to catalyse their oxidation less efficiently. We report a structure-guided kinetic and biophysical investigation of Arabidopsis thaliana PCO4 that reveals residues important for substrate-binding and catalysis. We subsequently selected At PCO4 variants Y183F and C173A, with severe and mild impacts on At PCO4 activity, respectively, to complement Arabidopsis pco1pco2pco4pco5 plants and investigate their impact on submergence resilience. Both variants appeared to be beneficial for survival and recovery after 2.5 and 3.5 days of dark submergence when compared to control plants, indicating that engineering PCOs can be used as a strategy to improve flood tolerance in plants.

Motivation Approximate string matching (ASM) is the problem of finding all occurrences of a pattern P in a text T while allowing up to k errors. ASM was researched extensively around the 1990s, but with the rise of large-scale datasets, focus shifted towards inexact approaches based on seed-chain-extend . These methods often provide large speedups in practice, but do not guarantee finding all matches with ≤ k errors. However, many applications, such as CRISPR off-target detection, require exhaustive results with no false negatives. Methods We introduce Sassy, a library and tool for ASM of short (up to ≈1000 bp) patterns in large texts. Sassy builds on earlier tools that use Myers’ bitpacking, such as Edlib. Algorithmically, the two main novelties are to split each sequence into 4 parts that are searched in parallel, and to use bitvectors in the text direction ( horizontally ) rather than the pattern direction ( vertically ). This allows significant speedups for short queries, especially when k is small, as has complexity O ( k⌈n/W⌉ ) when searching random text, where W = 256 is the SIMD width. Practically speaking, Sassy is the only recent index-free tool that is designed specifically for ASM, rather than the more common semi-global alignment. In addition, Sassy supports the IUPAC alphabet, which is essential for primer design and for matching ambiguous bases in assemblies. Separately, we also introduce the concept of overhang cost a variant of ‘overlap’ alignment where e.g. a suffix of the pattern is matched against a prefix of the text, where each character of the pattern that extends beyond the text incurs a cost of e.g. α = 0.5. This is important when matches are near contig or read ends. Results Compared to Edlib, Sassy is 4 × to 15 × faster for sequences up to length 1000, and has throughputs exceeding 2 GB/s, whereas Edlib remains below 130 MB/s. Likewise, Sassy is up to 10 × faster than parasail when searching short strings. Sassy is also readily applicable to biological problems such as CRISPR off-target detection. When searching 61 guide sequences in a human genome, Sassy is 100 × faster than SWOffinder and only slightly slower (for k ≤ 3) than CHOPOFF, for which building its index takes 20 minutes. Sassy also scales well to larger k ∈ {4, 5}, unlike CHOPOFF whose index took over 10 hours to build. Availibility Sassy is available as Rust library and binary at https://github.com/RagnarGrootKoerkamp/sassy .

Abstract Tomato high pigment-2 (hp-2dg, hp-2, and hp-2j) mutant lines are characterized by mutations in the DE-ETIOLATED1(SlDET1; Solyc01g056340) gene. SlDET1 is responsible for encoding a nuclear protein that acts as a negative regulator involved in light signaling, repressing photomorphogenesis. These tomato mutant lines are known for increased levels of antioxidant pigments in fruits, such as flavonoids and carotenoids, compared to the wild-type fruits. In this study, CRISPR/Cas9 followed by the non-homologous end joining mechanism of repair (NHEJ), was used to target the SlDET1gene and investigate whether the effects of these mutations could mimic the effects of hp-2 mutant lines, improving the nutritional features of tomato fruits. Our results indicated that mutations generated by CRISPR/Cas9 NHEJ in the hp-2and hp-2j regions (exon 11) resulted in significant changes in the SlDET1 coding and protein sequences, causing a low survival rate of edited sprouts and regenerated plants with very compromised capacity of allelic heritability of these mutations for the following generations. However, regenerated plants containing these site-specific mutations in the SlDET1 gene showed higher levels of phytochemicals in ripe fruits. Furthermore, these edited plants also showed an upregulation of structural genes involved in the synthesis of these biocompounds. Although the SlDET1 gene could be considered an interesting target gene for the nutritional improvement of tomato fruit, our results showed that mutations within its exon 11 are quite critical and can induce severe perturbations in plant metabolism, physiology, and survival, with a compromised possibility to develop new stable edited lines.

Abstract Corneal neovascularization is a sight-threatening condition for which current treatments such as anti-VEGF agents are limited by invasiveness and side effects. We present the first non-viral, CRISPR/Cas9-based gene therapy delivered via topical eye drops that penetrates the cornea and inhibits pathological neovascularization. Cas9 ribonucleoproteins (RNPs) targeting the Vegfa gene were complexed with a liposomal carrier (lipofectamine) and administered to mice after alkali burn injury to the cornea. This approach achieved approximately 2% gene editing at the Vegfa locus in vivo, which significantly reduced local VEGF-A expression. Consequently, treated corneas showed markedly decreased macrophage infiltration and robust suppression of both hemangiogenesis and lymphangiogenesis compared to untreated controls. These findings demonstrate that even modest in vivo gene editing can yield a strong therapeutic effect, highlighting a clinically relevant strategy for controlling corneal angiogenesis. Our study introduces a feasible and safe topical CRISPR therapy for corneal diseases, offering a potential alternative to invasive or virus-based gene delivery methods.

Background: Autoimmune diseases are chronic, debilitating conditions caused by the immune system mistakenly attacking healthy tissues. Conventional treatments mainly involve broad immunosuppression, which is associated with significant side effects, lim-ited specificity, and suboptimal long-term outcomes. Objective: This review aims to ex-plore recent advances in nanotechnology-based therapies for autoimmune diseases, fo-cusing on their mechanisms, therapeutic applications, and clinical translation potential. Methods: A comprehensive review of peer-reviewed literature was conducted to examine various nanotechnology platforms, including drug-loaded nanoparticles, antigen-specific nanomedicines, RNA interference (siRNA), CRISPR-enabled systems, and stimu-li-responsive nanocarriers. Key preclinical and clinical studies were highlighted to evalu-ate efficacy and safety. Results: Nanomedicine offers multiple advantages over traditional therapies, such as targeted drug delivery, improved bioavailability, reduced systemic tox-icity, and potential induction of immune tolerance. Notable innovations include biode-gradable polymeric nanoparticles, liposomes, micelles, exosome-mimetic nanoparticles, and magnetic nanomaterials. Emerging technologies like CRISPR-Cas9 and RNAi deliv-ered via nanoparticles are advancing immune modulation in autoimmune models. De-spite promising outcomes, several barriers remain, including toxicity concerns, scale-up manufacturing issues, and regulatory challenges. Conclusion: Nanotechnology is rede-fining autoimmune disease therapy by shifting from non-specific immunosuppression to precision-targeted approaches. Future progress lies in integrating nanomedicine with personalized medicine to tailor treatments based on individual immune profiles. Contin-ued interdisciplinary collaboration and regulatory alignment are essential to translating these innovations into clinical practice.

Chondrosarcoma is the most common type of primary bone sarcoma in adults but with a high risk of local recurrence and metastasis. Most chondrosarcomas are resistant to chemotherapy and radiotherapy, which means that surgery is the only effective treatment option for the majority of patients. Therefore, new therapeutic targets are required. CD44 is a transmembrane protein that has roles in cell proliferation, adhesion and migration and it has already been shown to be overexpressed in several cancer types. In this study, chondrosarcoma patient tissue sections were characterised for CD44 expression using immunohistochemistry. The relationship between CD44 expression in the patient tissue and overall and event-free survival was then investigated. CRISPR/Cas9 gene editing was also used to knockout CD44 from chondrosarcoma cells which were used in a spheroid invasion assay to understand the role of CD44 in chondrosarcoma cell invasion. Cox multivariate analysis of CD44 expression by chondrosarcoma patient tissue revealed that high CD44 expression was linked to decreased overall and event-free survival. Furthermore, the invasion of the CD44 knockout cells in the spheroid invasion assay was less than the invasion of the wild-type cells. Increased expression of CD44 for intermediate and high grades of chondrosarcoma as well as reduced invasion of CD44 knockout cells suggests that CD44 plays an important role in chondrosarcoma metastasis. CD44 is therefore worthy of further investigation as an imaging and therapeutic target.

SUMMARY The SAGA transcriptional co-activator complex regulates gene expression through histone acetylation at promoters, mediated by its histone acetyl transferase, KAT2A. While its structure and function have been extensively investigated, how the stability of individual subunits of SAGA, including KAT2A, is regulated, remains unclear. Here, using a fluorescence-based KAT2A stability reporter, we systematically dissect the molecular dependencies controlling KAT2A protein abundance. We identify the non-enzymatic SAGA CORE module subunits—TADA1, TAF5L, and TAF6L— as necessary for KAT2A stability, with loss of these subunits disrupting the integrity of SAGA, leading to non-chromatin-bound KAT2A that is degraded by the proteasome, consequently leading to reduced H3K9 acetylation. Proteomic profiling reveals progressive loss of CORE and HAT components upon acute disruption of the SAGA CORE, indicating that an intact CORE is required for the stability of numerous components of SAGA. Finally, a focused CRISPR screen of ubiquitin-proteasome system genes identifies the E3 ligase UBR5, a known regulator of orphan protein degradation, and the deubiquitinase OTUD5, as regulators of KAT2A degradation when the SAGA CORE is perturbed. Together, these findings reveal a dependency of KAT2A protein stability on SAGA CORE integrity and define an orphan quality control mechanism targeting unassembled KAT2A, revealing a potential vulnerability in SAGA-driven malignancies.

High-throughput profiling of neuronal activity at single-cell resolution is essential for advancing our understanding of brain function, enabling large-scale functional screens, and modeling neurological disorders. However, existing approaches are limited by scalability, manual data processing, and variability, thus restricting their ability to detect disease-associated phenotypes. Here, we present a scalable, open-source platform that integrates optogenetic stimulation, calcium imaging, automated data acquisition, and a fully integrated analysis pipeline. By combining spontaneous and evoked activity profiling, the system enables robust quantification of dynamic neuronal responses across hundreds of stem cell-derived human neurons and multiple timepoints, facilitating phenotyping at both cellular and network levels. We validated the platform by recapitulating established activity phenotypes in neurodevelopmental disorders including CDKL5 Deficiency Disorder and SSADH deficiency. In addition, we generated CRISPR-Cas9 knock-in human induced pluripotent stem cell (hiPSC) lines stably expressing the genetically encoded calcium indicator GCaMP6s to model network dysfunction in Tuberous Sclerosis Complex (TSC). Using this system, we further demonstrated functional rescue of the altered neuronal activity observed in the TSC following pharmacological intervention. By linking single-cell dynamics to population-level phenotypes, this framework provides a powerful and broadly applicable tool for disease modeling, mechanistic studies, and therapeutic screening across a range of neurological disorders.

Elucidating the spatial and temporal regulation of gene expression during plant organogenesis is crucial for enabling precise crop improvement strategies that incorporate beneficial traits into crops while avoiding adverse effects. Root nodules, specialised organs formed in symbiosis with nitrogen-fixing bacteria, provide a valuable system to study cell-type-specific gene networks in a symbiosis-induced developmental context. However, capturing these dynamics at cellular resolution in intact plant tissues remains technically challenging. Spatial transcriptomics technologies developed for animal systems are often not directly transferable to plant tissues due to fundamental differences in tissue composition between plants and animals, including rigid and heterogeneous plant cell walls, high cell wall autofluorescence, and large vacuoles in plant cells that complicate probe access and signal detection. To address these challenges, we present an optimised protocol for applying the Xenium in situ sequencing platform to formalin-fixed paraffin-embedded (FFPE) sections of plant tissues, including Medicago truncatula roots and nodules. Key technical adaptations include customised tissue preparation, optimised section thickness, hybridisation conditions, post-Xenium staining, imaging, and downstream image analysis, all tailored specifically for plant samples. To mitigate autofluorescence and enhance detection sensitivity, we employed a strategic approach to codeword selection during probe design. Furthermore, we developed a modular probe design approach combining a custom 380-gene standalone panel with a 100-gene add-on panel. This design allows flexibility for addressing diverse research questions and includes orthologous gene sequences from two Medicago ecotypes, ensuring compatibility for downstream functional validation using mutant lines available in both genetic backgrounds. We validated the protocol across nodules at multiple developmental stages using both the 50-gene panel targeting mature nodule cell identity and the extended 480-gene panel, which includes markers across different cell types and developmental stages, as well as genes of interest identified from prior single-cell and bulk RNA-seq analyses. This optimised workflow provides a reproducible and scalable method for high-resolution spatial transcriptomics in plant tissues, establishing a robust foundation for adaptation to other plant species and developmental systems.

Extracellular vesicles (EVs) are crucial mediators of intercellular communication that originate through one of two pathways: outward budding of the plasma membrane (PM) or fusion of mature (late) endosomes with the PM. How cells balance these EV biogenesis routes remains unclear. To address this, we performed a genome-wide CRISPR activation screen for genes that increase cell surface levels of the tetraspanin CD63—an EV marker that shuttles between late endosomes and the PM. This unbiased approach identified a membrane adaptor protein, MARCKSL1, commonly upregulated in diverse tumor types. Follow-up studies, integrating genomic activation/ablation with microscopic and proteomic approaches, revealed that MARCKSL1 potentiates EV secretion from the PM, (in part) at the expense of late endosome—PM fusion. Probing the molecular context of MARCKSL1 function, we implicate PM-bridging cytoskeletal components (e.g., Radixin) and SNARE-associated proteins (e.g., STXBP3) as collaborators of MARCKSL1. Collectively, our findings reveal new mechanistic underpinnings of PM remodeling and position MARCKSL1 as a gauge between different platforms of EV biogenesis.

Photosynthetic organisms have evolved mechanisms to manage excess light, crucial for maximizing photosynthetic efficiency. High-light (HL) tolerant Synechocystis sp. PCC6803 strains were developed through laboratory evolution, with tolerance attributed to specific point mutations. Key mutations affected the NDH-1L complex F1-subunit (NdhF1 F124L ) and translation elongation factor G2 (EF-G2 R461C ). Reintroducing these mutations into laboratory strains conferred HL tolerance. Comparisons with knockout and overexpressor lines showed NdhF1 F124L and EF-G2 R461C result in gain of function. Transcriptomic and proteomic analysis unveiled a network of responses contributing to HL tolerance, including maintenance of phosphate metabolism and decreased antenna size by depleting a specific linker protein in EF-G2 R461C cells. Consequently, overexpression of Pho regulon genes increased HL tolerance. NdhF1 F124L enhances cyclic electron flow (CEF) by increasing NDH-1 complex subunit accumulation. Other HL-adapted strains demonstrated that increased CEF and decreased antenna size are recurring outcomes, achievable through various mutations. This study demonstrates how limited mutations can reconfigure cells for enhanced HL tolerance, offering insights for improving photosynthetic efficiency.

ZIC2, a member of the Zinc Finger of the Cerebellum family of transcription factors (TFs), plays crucial roles during neural development. In humans, defects in ZIC2 cause holoprosencephaly, a congenital brain malformation characterized by the defective cleavage of cerebral hemispheres due to problems in midline patterning. However, the gene regulatory network (GRN) controlled by ZIC2 and the regulatory mechanisms it employs during neural development remain largely unexplored. Here, we combined a mouse embryonic stem cell (mESC) in vitro differentiation model towards anterior neural progenitors (AntNPCs) with genome editing approaches, bulk and single cell (i.e. Multiome scATAC + scRNAseq) genomic methods to elucidate the precise GRN controlled by ZIC2 and the underlying mechanisms. We found that ZIC2 shows widespread binding throughout the genome already in mESC as well as upon pluripotency exit and neural induction. Despite its extensive binding in mESC, ZIC2 function is dispensable in pluripotent cells due to compensation by ZIC3. In contrast, ZIC2 plays a major regulatory function during neural induction, directly controlling the expression of master regulators implicated in the patterning and morphogenesis of specific brain regions, such as the midbrain (e.g., En1, Lmx1b, Pax2, Wnt1 ) and the roof plate (e.g., Lmx1a, Wnt3a ). Mechanistically, ZIC2 plays a dual role in neural differentiation: (i) during pluripotency exit, ZIC2 acts as a pioneer TF, binding de novo to distal enhancers and promoting their chromatin accessibility; (ii) during neural induction, ZIC2 is essential for the activation of a subset of the previously primed enhancers, which in turn control the expression of major neural patterning regulators and signaling pathways (i.e. WNT) that prevent the premature differentiation of neural progenitors. Overall, our work shows that, by sequentially acting as a promiscuous pioneer and selective activator of enhancer elements, ZIC2 canalizes pluripotent cells towards neural progenitors with rostro-dorsal identities.

DNA lesions block the progression of RNA polymerase II (RNAPII) during transcription, impeding gene expression and threatening genome integrity. When RNAPII stalls on transcription-blocking lesions, the transcription-coupled DNA repair pathway is activated to remove the DNA damage. Following DNA repair, efficient transcription restart depends on the PAF1 elongation complex (PAF1C). PAF1C contributes to deposition of transcription-associated histone marks, including H2B-K120 Ub , H3K4me 3 and H3K79me 2 . These marks are enriched at actively transcribed genes and have been associated with regulation of post-repair transcription restart. Here, we show that the H2B-K120 E3 ubiquitin ligase RNF20/RNF40, the H3K4-methyltransferase SET1/COMPASS complex, and the H3K79-methyltransferase DOT1L are dispensable for transcription restart. Moreover, levels of H2B-K120 Ub and H3K4me 3 do not correlate with transcription restoration following DNA damage. Additionally, we observe that, unlike PAF1, the dissociable PAF1C subunit RTF1, while stimulating H2B-K120 Ub and H3K4me 3 , does not play a role in transcription restart. Together, these data suggest that transcription restoration after DNA damage is stimulated by the PAF1C elongation complex, independently of transcription-associated histone mark deposition.

Summary Multipolar migration is a conserved neuronal migration mode in the developing brain, enabling emerging neurons to navigate crowded environments and reach precise laminar positions. Yet how these cells interpret external cues to guide their migration remains unclear. We investigate this question in the developing vertebrate retina using horizontal cells as a model. Combining transcriptomics, targeted CRISPR screening, and live imaging, we reveal the spatiotemporal guidance system underlying horizontal cell lamination: repulsive Slit1b/2–Robo2 signaling in the amacrine cell layer is essential to initiate apical horizontal cell migration, while attractive Neurturin–Gfrα1 signaling from photoreceptors fine-tunes final positioning beneath the photoreceptor layer. Disruption of these pathways causes basal retention of horizontal cells, highlighting the importance of spatially coordinated signaling for proper lamination and functional retinal circuitry. Our results uncover how positional signals and tissue architecture cooperate to achieve neuronal precision, an organizing principle likely relevant across the developing central nervous system.

The restoration of uniformly-distributed dystrophin protein expression is an important consideration for the development of advanced therapeutics for Duchenne muscular dystrophy (DMD). To explore this concept, we generated a novel genetic mouse model ( mdx52-Xist Δhs ) that expresses variable, and non-uniformly distributed, dystrophin protein from birth as a consequence of skewed X-chromosome inactivation. mdx52-Xist Δhs myofibers are heterokaryons containing a mixture of myonuclei expressing either wild-type or mutant dystrophin alleles in a mutually exclusive manner, resulting in dystrophin protein being spatially restricted to corresponding dystrophin-expressing myonuclear domains. This phenotype models the situation in female DMD carriers, and dystrophic muscle in which dystrophin has been incompletely restored by partially-effective experimental therapeutics. Total dystrophin expression increased in aged (60-week-old) mdx52-Xist Δhs mice relative to 6-week-old adults, suggestive of an accumulation of dystrophin-expressing myonuclei through positive selection, although this was insufficient to resolve sarcolemmal dystrophin patchiness. Nevertheless, compared to mice expressing no dystrophin, non-uniformly-distributed dystrophin was protective against pathology-related muscle turnover in an expression-level-dependent manner in both adult and aged mdx52-Xist Δhs mice. Systematic classification of isolated mdx52-Xist Δhs myofibers revealed profound differences associated with central nucleation, with dystrophin found to be translationally repressed in centrally-nucleated myofibers and myofiber segments. These findings have important implications for the development of dystrophin restoration therapies.

ABSTRACT Disease-associated variants can lead to variable phenotypic outcomes, but the biological mechanisms underlying this variability remain poorly understood. We developed a framework to investigate this phenomenon using the 16p12.1 deletion as a paradigm of variable expressivity. Using induced pluripotent stem cell models from affected families and CRISPR-edited lines with the 16p12.1 deletion, we found that the deletion and secondary variants in the genetic background jointly influenced chromatin accessibility and expression of neurodevelopmental genes. Cellular analyses identified family-specific phenotypes, including altered inhibitory neuron production and neural progenitor cell proliferation, which correlated with head-size variation. CRISPR activation of individual 16p12.1 genes variably rescued these defects by modulating key signaling pathways such as TGF-β and PI3K-AKT. Integrative analyses further identified regulatory hubs, including transcription factors FOXG1 and JUN, as mediators of these effects. Our study provides a functional framework for investigating how individual genetic architectures contribute to phenotypic variability in genetic disorders.

Symbiotic nitrogen fixation (SNF) is a key trait in legume productivity, yet the genetic and regulatory basis underlying its natural variation remains poorly understood. Here, we integrated genome, transcriptome, and chromatin accessibility data from a soybean diversity panel comprising 380 accessions, including 108 wild and 272 cultivated lines. Genome-wide association studies (GWAS) detected multiple loci for SNF traits but with limited resolution due to polygenic architecture and environmental influences. Independent component analysis (ICA) identified 136 co-expression modules; ten ICs were strongly correlated with SNF phenotypes and enriched in circadian clock components (e.g., GmLHY1a/b), lipid metabolism, or defense signaling pathways. Transcriptome-wide association studies (TWAS) linked 1,453, 806, and 178 genes to NFP, NW, and NFE traits, respectively. Among TWAS hits, 185 transcription factors were identified, with 39.0% overlapping selective sweeps, suggesting regulatory evolution under domestication. To further dissect expression regulation, we performed eQTL mapping and detected 4,654 significant eQTLs, including 1,241 local (cis), 2,505 distal (trans), and 908 mixed. By integrating ATAC-seq data from sorted nodule nuclei, we found that eQTLs, particularly local eQTLs, are significantly enriched within open chromatin regions, indicating their regulatory potential. Notably, we validated the circadian clock gene GmLHY1b as a negative regulator of nodulation using CRISPR mutagenesis and CUT&Tag. Our integrative study provides comprehensive genomic and transcriptomic resources from a diverse soybean population, offering novel insights into SNF regulatory networks and a valuable foundation for future SNF research and soybean improvement.

Abstract Lignocellulosic biorefining has traditionally focused on either converting biomass into sugars for fuels or isolating solid cellulose for bioproducts. However, cost-effective strategies to maximize sugar yields while preserving crystalline cellulose remain underexplored. This study addresses that gap by optimizing cellulase enzymes to the co-production of fermentable sugars and crystalline cellulose. Laboratory-scale results also informed a techno-economic analysis (TEA) to evaluate the feasibility of industrial-scale implementation. To this end, we developed a selective hydrolysis process that targets hemicelluloses and amorphous cellulose, while retaining crystalline regions, using an optimized enzyme cocktail tested on three feedstocks: unbleached hardwood pulp, wild-type poplar, and clustered regularly interspaced short palindromic repeats (CRISPR)-edited poplar. Optimization across varying pH and temperature conditions enabled effective selective hydrolysis. Low-lignin unbleached pulp and CRISPR-edited poplar exhibited improved enzymatic accessibility and required less pretreatment, resulting in higher sugar yields and more efficient downstream processing. An engineered yeast strain co-fermented C5 and C6 sugars into ethanol, leaving behind high-crystallinity cellulose. CRISPR-edited poplar outperformed wild type, with 18% more sugar and 25% more ethanol yield, while enhancing cellulose crystallinity. TEA estimated crystalline cellulose production costs at $4,438 per metric tonne from unbleached pulp and $1,474 from CRISPR-edited biomass, highlighting the economic advantage of engineered feedstocks. This work presents a novel lignocellulosic biorefining approach that, for the first time, prioritizes the co-production of fermentable sugars and crystalline cellulose from low-lignin biomass.

Abstract Background Adoptive cell therapy (ACT) with genetically engineered T cells expressing chimeric antigen receptors (CARs) has emerged as a promising treatment option for patients with refractory leukaemia or lymphoma. Despite its success in type B malignancies, CAR-T cell therapy still faces some challenges such as toxicity, functional suppression by the tumour microenvironment (TME), and poor persistence in treated patients. Methods This study employed a second-generation CD19-targeting CAR construct to generate engineered CAR-T cells with enhanced functionality through precise genome editing. Using CRISPR/Cas9 technology, the PDCD1 gene was to mitigate T cell exhaustion, and in a parallel knock-in strategy, an IL-15 transgene was inserted at the PDCD1 locus. Gene editing was performed via electroporation of RNP complexes, with AAV6 vectors used for homology-directed IL-15 integration. Editing efficiency and off-target activity were assessed by flow cytometry, Sanger sequencing, ICE, and CAST-Seq. Functional characterization included bulk RNA sequencing, metabolic profiling using Seahorse technology, and cytotoxicity assays against CD19 + target cells. Results We initially demonstrated that αCD19 CAR-T cells lacking PD-1 expression (PD-1 KO) exhibited reduced expansion capacity and overall fitness compared to control CAR-T cells but showed a superior cytotoxicity against PDL1 + target cells. To address the impaired fitness of PD-1 KO CAR-T cells, we generated PD-1KIL-15 CAR-T cells, which combine PD-1 KO with the expression of IL-15 under the control of the PD-1 endogenous promoter. Compared to CAR T PD-1 KO cells, PD-1KIL-15 CAR-T cells displayed improved phenotype, viability, and metabolism. More importantly, they also demonstrated enhanced cytolytic capacity of PDL1 + CD19 + target cells, which correlated with increased resistance to apoptosis and improved cell fitness. Conclusions In summary, we present a next 4th generation CAR-T cells platform (TRUCKs) that integrates PD-1 deletion with the inducible expression of IL-15 upon T cell activation and/or exhaustion. This strategy addresses the limitations associated with knocking-out PD-1 and those associated with sustained IL-15 cytokine expression. The same platform can be used to generate PD-1 KO TRUCKs targeting different antigens and expressing different cytokines under the control of the PD-1 locus.

SUMMARY SnRK1 is an evolutionarily conserved protein kinase belonging to SNF1/AMPK family of protein kinases that is central to adjusting growth in response to the energy status. Numerous studies have shown adaptive and developmental roles of SnRK1, but the understanding of SnRK1 signaling network in monocots is limited. Using CRISPR/Cas9 mutagenesis to target the functional kinase subunits in rice, we carried out a comprehensive phenotypic, transcriptomic, proteomic, and phosphoproteomic analyses of rice snrk1 mutants displaying growth defects under normal and starvation conditions. These analyses revealed the role of SnRK1 signaling in controlling growth and stress-related processes in both energy-sufficient and energy-limited conditions and pointed to sub-functionalization of SnRK1 kinase subunit genes. In addition to the classical protein targets of SnRK1, phosphoproteomics revealed novel targets including the key components of intracellular membrane trafficking, ethylene signaling, and ion transport. The upregulation of stress-related processes and suppression of growth-related processes in snrk1 mutants correlated with their phenotypic defects. Overall, this study highlights the dual role of SnRK1 as promoter of growth under favorable conditions and critical regulator of adaptive response under stress conditions.

Human papillomavirus (HPV) plays a major role in the development of head and neck cancers (HNCs), particularly oropharyngeal squamous cell carcinoma. This review highlights the key molecular mechanisms of HPV-driven carcinogenesis, focusing on the oncogenic E6 and E7 proteins and their disruption of tumor suppressor pathways and epigenetic regulation. We discuss the rising prevalence of HPV-related HNCs, their distinct clinical features, and diagnostic approaches such as p16 immunohisto-chemistry and HPV DNA/RNA detection. HPV-positive tumors show better prognosis and response to treatment, prompting interest in therapy de-escalation. Emerging strategies including immune checkpoint inhibitors, therapeutic vaccines, CRISPR-based gene editing, and ctDNA monitoring are advancing precision oncology in this field. We also examine the preventive potential of HPV vaccination and ongoing research into its role across various HNC subtypes. A deeper understanding of HPV’s molecular impact may guide more effective, targeted, and less toxic interventions.