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.

Genetic studies of human embryonic morphogenesis are constrained by ethical and practical challenges, restricting insights into developmental mechanisms and disorders. Human pluripotent stem cell (hPSC)–derived organoids provide a powerful alternative for the study of embryonic morphogenesis. However, screening for genetic drivers of morphogenesis in vitro has been infeasible due to organoid variability and the high costs of performing scaled tissue-wide single-gene perturbations. By overcoming both these limitations, we developed a platform that integrates reproducible organoid morphogenesis with uniform single-gene perturbations, enabling high-throughput arrayed CRISPR interference (CRISPRi) screening in hPSC-derived organoids. To demonstrate the power of this platform, we screened 77 transcription factors in an organoid model of anterior neurulation to identify ZIC2 , SOX11 , and ZNF521 as essential regulators of neural tube closure. We discovered that ZIC2 and SOX11 are required for closure, while ZNF521 prevents ectopic closure points. Single-cell transcriptomic analysis of perturbed organoids revealed co-regulated gene targets of ZIC2 and SOX11 and an opposing role for ZNF521 , suggesting that these transcription factors jointly govern a gene regulatory program driving neural tube closure in the anterior forebrain region. Our single-gene perturbation platform enables high-throughput genetic screening of in vitro models of human embryonic morphogenesis.

Background Idiopathic pulmonary fibrosis is a fatal lung disease of progressive lung parenchymal scarring caused by the aberrant response of an alveolar epithelium repeatedly exposed to injury. Understanding epithelial dysfunction has been hampered by the lack of physiological alveolar type 2 (AT2) cell models and defined disease triggers. Monogenic forms of familial pulmonary fibrosis (FPF) caused by toxic gain-of-function variants provide an opportunity to investigate early pathogenic events. One such variant, surfactant protein C (SFTPC)-I73T, abnormally localises within AT2 cells and causes their dysfunction. Methods We used base editing of fetal lung-derived AT2 (fdAT2) organoids to create a heterozygous disease model of endogenous SFTPC-I73T expression. We also created an inducible overexpression system to interrogate temporal changes associated with SFTPC-I73T expression. We cultured fdAT2 both in 3D culture and at air-liquid interface to understand the importance of polarity cues and air exposure on disease phenotypes. Results In our heterozygous endogenous expression system, we found that fdAT2 expressing SFTPC-I73T grew without a lumen and were unable to correctly polarise. SFTPC-I73T accumulated with time and caused gross enlargement of early endosomes, preventing correct apico-basal trafficking of multiple endosomally trafficked cargoes including polarity markers and cell adhesion proteins. This phenotype was exacerbated by air exposure and led to loss of epithelial monolayer integrity and abnormal wound healing after injury. Conclusion Using endogenous gene editing for the first time in differentiated alveolar organoids, we have demonstrated that the pathogenic effects of SFTPC-I73T are mediated through endosomal dysfunction and abnormal epithelial organisation. This has important implications for AT2 function in vivo .

The establishment of the body plan during gastrulation represents a hallmark of animal life. It emerges from the interplay of gene-regulatory programs and positional cues, yet how these signals are integrated post-transcriptionally remains largely unexplored. Here, we combine the scalability of mouse gastruloids with a single-cell CRISPR screening platform to functionally dissect germ layer specification at single-cell transcriptomic resolution. Focusing on post-transcriptional regulation, we systematically map drivers of mesodermal and endodermal fate and identify the deadenylase Cnot8 . Loss of Cnot8 leads to widespread poly(A) tail elongation and transcript stabilization, shifting mesoderm differentiation toward ectopic notochord fate, thereby profoundly impacting axial patterning. Collectively, our findings identify mRNA deadenylation as a fundamental mechanism linking cellular identity with morphogenetic signaling during mammalian body plan formation.

Streptococcus mutans is recognized as the primary etiological agent of dental caries, one of the most prevalent infectious diseases globally. Its remarkable acid tolerance enables survival and proliferation in the low-pH biofilm microenvironment, establishing S. mutans as the dominant species in dental plaque and a key contributor to cariogenesis. Although numerous studies have identified genes linked to acid tolerance mechanisms, the full set of essential acid tolerance genes within its genome remains incompletely characterized, largely due to the lack of systematic, genome-scale investigations. To address this knowledge gap, we constructed a genome-wide pooled CRISPR interference (CRISPRi) library targeting 95% of the predicted S. mutans genes and employed next-generation sequencing to identify acid tolerance determinants systematically. Our screen revealed 95 acid tolerance-associated genes, a subset of which were functionally validated through gene knockout studies. Functional enrichment analysis demonstrated significant associations with metabolic pathways (including cofactor biosynthesis and amino/nucleotide sugar metabolism), tRNA modification, and transcriptional regulation. Protein-protein interaction (PPI) network analysis identified critical interactors (ComYC, SMU_1979c, DeoC, AcpP, NadD, and SMU_1988c) and two functionally cohesive modules. These findings provide novel mechanistic insights into the acid adaptation strategies of S. mutans and highlight potential therapeutic targets for caries prevention.

CRISPR activation (CRISPRa) enables precise, locus-specific upregulation of gene expression, offering potential for both ex vivo and in vivo applications. However, the lack of scalable, high-coverage tools has limited its use in comprehensive genetic screens, particularly in murine models. Here, we introduce Partita, a next-generation, whole-genome CRISPRa sgRNA platform designed for unparalleled efficiency and depth in gene activation studies. Partita employs a high-density targeting strategy, deploying 10 sgRNAs per transcription start site, structured into five gene family-specific sub-libraries to maximize transcriptional induction. To demonstrate its capabilities, we performed a series of large-scale screens: an in vitro enrichment/depletion screen in iBMDMs, whole-genome CRISPRa screens in a double-hit lymphoma model to uncover genes driving resistance to pro-apoptotic drugs (venetoclax, nutlin-3a, etoposide), and an in vivo whole-genome screen identifying accelerators of Myc-driven lymphomagenesis. Each experiment revealed both expected and novel regulators of cellular phenotypes, with a high validation rate in secondary assays. By enabling robust, high-throughput gain-of-function screening, Partita unlocks new avenues for functional genomics and expands the toolkit for discovering key drivers of biological processes across diverse research fields.

Clustered regularly interspaced short palindromic repeat Cas endonuclease (CRISPR-Cas) systems, such as RNA-editing CRISPR-Cas13d, are poised to advance the gene therapy of various diseases. However, their clinical development has been challenged by 1) the limited biostability of linear guide RNAs (lgRNAs) susceptible to degradation, 2) the immunogenicity of prokaryotic microorganism-derived Cas proteins in human that restrains their long-term therapeutic efficacy, and 3) off-targeting gene editing caused by the prolonged Cas expression from DNA vectors. Here, we report the development of highly stable circular gRNAs (cgRNAs) and transiently-expressing Cas13d-encoding mRNA for efficient CRISPR-Cas13d editing of target mRNA. We first optimized cgRNA for CRISPR-Cas13d editing of adenosine deaminase acting on RNA type I ( Adar1 ) transcript for the combination immunotherapy of triple negative breast cancer (TNBC). cgRNAs were synthesized by enzymatic ligation of lgRNA precursors. cgRNAs enhanced biostability with comparable Cas13d-binding affinity relative to lgRNA. Next, using ionizable lipid nanoparticles (LNPs), we co-delivered the resulting Adar1 -targeting cgRNA with an mRNA encoding RfxCas13d (mRNA-RfxCas13d), a widely used Cas13d variant, to TNBC cells. As a result, relative to lgRNA, cgRNA significantly enhanced the efficiency of Adar1 knockdown with minimal collateral activity, which sensitized the cancer cells for cytokine-mediated cell apoptosis. In a 4T1 murine TNBC tumor model in syngeneic mice, Adar1 -targeting cgRNA outperformed lgRNA for tumor immunotherapy in combination with immune checkpoint blockade (ICB). Collectively, these results demonstrate the great potential of cgRNA and mRNA-RfxCas13d for RNA-targeted gene editing.

Soil salinity varies widely across geographies both due to natural factors and human activities, including agriculture, road salt application, sea level rise, and desertification. Increases in soil salinity may affect organisms widely and particularly impact soil foodwebs. As parasites, entomopathogenic nematodes (EPNs) occupy crucial links in soil foodwebs and are important for agriculture as biological control agents of insect pests. Previous research found that the EPN Steinernema carpocapsae may exhibit higher salt tolerance than several of its congeners. We recently identified that S. carpocapsae uniquely evolved two amino acid substitutions in the first extracellular loop of the sodium pump (Na□/K□-ATPase). Here, we tested if these substitutions explain S. carpocapsae ’s reported lower sensitivity to salt. Our results confirm that S. carpocapsae exhibits higher salt tolerance and show it can more effectively locate and infect insect hosts than its congeners S. feltiae and S. hermaphroditum in highly saline environments. We then retraced the evolution of the two amino acid substitutions in S. carpocapsae by introducing them alone and in combination in Caenorhabditis elegans using CRISPR genome engineering. We found that C. elegans mutants with single substitutions showed improved salt tolerance. However, this improvement disappeared in the double mutant, whose sodium pump mimicked that of S. carpocapsae . This pattern of negative epistasis between the amino acid substitutions suggests they are not responsible for variation in salt tolerance between Steinernema species. Sodium pump evolution in S. carpocapsae might instead be driven by encounters with cardiac glycosides, which are released into soil by several clades of plants including milkweeds, sequestered by some of this EPN’s herbivorous insect hosts, and known to target the first extracellular loop of the sodium pump. Our findings provide valuable insights into EPN adaptation to changes in environmental sodium levels and may have implications for their use in biological control.

ABSTRACT Bacterial flagella drive motility and chemotaxis while also playing critical roles in host-pathogen interactions, as their oligomeric subunit, flagellin, is specifically recognized by the mammalian immune system and flagellotropic bacteriophages. We recently discovered a family of phage-encoded, RNA-guided transcription factors known as TldR that regulate flagellin expression. However, the biological significance for this regulation, particularly in the context of host fitness, remained unknown. By focusing on a human clinical Enterobacter isolate that encodes a Flagellin Remodeling prophage (FRφ), here we show that FRφ exploits the combined action of TldR and its flagellin isoform to dramatically alter the flagellar composition and phenotypic properties of its host. This transformation has striking biological consequences, enhancing bacterial motility and mammalian immune evasion, and structural studies by cryo-EM of host- and prophage-encoded filaments reveal distinct architectures underlying these physiological changes. Moreover, we find that FRφ improves colonization in the murine gut, illustrating the beneficial effect of prophage-mediated flagellar remodeling in a host-associated environment. Remarkably, flagellin-regulating TldR homologs emerged multiple times independently, further highlighting the strong selective pressures that drove evolution of RNA-guided flagellin control. Collectively, our results reveal how RNA-guided transcription factors emerged in a parallel evolutionary path to CRISPR-Cas and were co-opted by phages to remodel the flagellar apparatus and enhance host fitness.