ID: 41416641
Title: The road to restore male fertility using in vitro-derived germ cells.
Abstract: Human-induced pluripotent stem cells (hiPSCs) offer immense potential in reproductive medicine, particularly for males who lack germ cells and cannot achieve biological parenthood through conventional ARTs. Early efforts to derive human germ cells from stem cells were hindered by low efficiency, subpar characterization, and the lack of standardized differentiation approaches. However, recent advancements have led to the development of defined protocols that mimic early embryonic development and allow the specification of transcriptomically and epigenetically validated human primordial germ cell-like cells (hPGCLCs). Current research focuses on maturing hPGCLCs in vitro, particularly within 3D culture systems that resemble their physiological microenvironment, with the aim of producing transplantable hiPSC-derived spermatogonial stem cells (SSCs) or differentiating them to sperm. At the same time, researchers are also testing whether hiPSCs generated from infertile patients can resume germline differentiation. This narrative review aimed to summarize the key efforts and remaining challenges in differentiating male germ cells from human pluripotent stem cells (hPSCs), with a particular focus on defined and validated protocols for hPGCLC specification. In parallel, we addressed key safety and ethical considerations that must be accounted for the development of clinical applications. A deeper understanding of the approaching therapeutic use of hiPSCs in reproductive medicine is essential for developing novel regenerative fertility strategies. PubMed, Scopus, and Web of Science were searched for studies attempting germ cell differentiation from hPSCs using relevant keywords ('stem cells', 'human pluripotent stem cells', 'human embryonic stem cells', 'human induced pluripotent stem cells', 'somatic cell reprogramming', 'infertility', 'germline', 'spermatogenesis', 'germ cells', and 'primordial germ cells'). No time period restriction was established. Studies with an exclusive focus on female germline differentiation were excluded. To maintain a human-focused perspective, only key animal studies are presented. The literature reveals a clear segregation among protocols for deriving germ cells from hPSCs, particularly between earlier studies lacking standardized differentiation conditions and characterization, and the most recent, defined protocols having transcriptomic and epigenetic validation against in vivo hPGCs. Moreover, during the last decade, the field has seen remarkable progress, with multiple efforts aimed at maturing hPGCLCs, closely recapitulating late male embryonic germline development. Additionally, hiPSCs derived from male patients at risk of infertility, particularly those without underlying genetic syndromes, generally retain the capacity for early germline commitment. While attempts at maturating patient germ cells beyond the hPGCLC state remain limited, the rapid pace of discovery and refinement in recent years suggests that further breakthroughs, including clinically applicable fertility restoration strategies, are likely to be achieved in the near future. The ability to generate hiPSCs from infertile patients and to specify them into hPGCLCs supports the feasibility of obtaining hiPSC-derived SSCs for future therapeutic use. These advances raise important ethical, regulatory, and societal questions that must be actively discussed among researchers, clinicians, policymakers, and the general public to ensure responsible and equitable access to these technologies. N/A.

ID: 41483428
Title: TNFRSF13B Variant-Induced TACI Dysregulation Underlies CAEBV Pathogenesis.
Abstract: The tumor necrosis factor (TNF) receptor superfamily member, transmembrane activator and CAML interactor (TACI) encoded by TNFRSF13B, are extensively involved in immune responses. In our previous work, TNFRSF13B exon 2 variants were recurrently identified in chronic active Epstein-Barr virus disease (CAEBV). Here we aim to reveal the roles of TNFRSF13B variants in CAEBV, and investigate the feasibility of targeting TNFRSF13B/TACI as a new approach to control EBV infection. The lymphoblastoid cell lines (LCL) models carrying homozygous TNFRSF13B exon 2 frameshift mutations were constructed using CRISPR/Cas9. Immunological assays, transcriptomic analysis, and gene silencing experiments were performed on LCL models to measure the effect of TNFRSF13B exon 2 variants and explore the underlying mechanisms. TACI ligands and a TLR9 agonist were applied to modulate TACI signaling and EBV activities. Frameshift mutations in exon 2 of TNFRSF13B significantly up-regulated the short isoforms of TACI (TACI-S) at the expense of its long isoforms (TACI-L) in LCLs. The up-regulated TACI-S induced more intense activation of NF-κB, MAPK, and Rho signaling pathways, leading to the switch of EBV activities to lytic reactivation. The subsequent increased viral load and viral IL-10 provide a rational for the susceptibility of variant carriers to CAEBV. The BAFF trimer, an indirect TACI-signaling inhibitor, also significantly suppressed the EBV lytic program. Gene silencing experiments indicated that XBP-1 might be involved in the TACI-mediated regulation of EBV lytic activities in EBV-immortalized B cells. This study underscores the impact of TNFRSF13B variants on EBV infection and host immune responses, offering insights into CAEBV pathogenesis and potential therapeutic strategies.

ID: 41581067
Title: Bioethics in assisted reproduction and embryology.
Abstract: The use of assisted reproductive therapies, advances in embryo research, and developments in scientific fields such as gene editing and in vitro gametogenesis have attracted the attention of bioethicists for years. On one side, the 14-day rule has faced criticism from embryo researchers advocating for its extension. On the other side, the increasing number of cryopreserved embryos worldwide has raised practical and ethical concerns about their fate. While advancements in scientific research, especially gene editing and in-vitro gametogenesis (IVG), are not yet fully applicable, their potential future use appears to pose significant bioethical questions. In this review, we examine the evolving bioethics of embryo research, focusing on the 14-day rule, the challenges surrounding surplus cryopreserved embryos, and the future dilemmas posed by CRISPR-based gene editing, IVG, and preimplantation genetic testing for polygenic risk (PGT-P). We also highlight the critical role of multidisciplinary, patient-centered counseling in ART practice, to foster informed consent, realistic expectations, and psychosocial well-being. Finally, we underscore the need for anticipatory ethical frameworks and open-society engagement that integrate public deliberation with scientific progress to ensure that reproductive innovation proceeds responsibly, preserving both human dignity and social justice.

ID: 41593679
Title: Adaptation of lentiviral vectors for viral gene therapy and their impact on host cell biology.
Abstract: Lentiviral vectors (LVVs) are used as a viral gene therapeutic and were derived from human immunodeficiency virus subtype 1 (HIV-1). LVVs are used to deliver and induce the stable expression of transgenes through genome integration. Current clinical LVV delivery systems do not include HIV-1 major accessory genes; however, critical structural and non-structural HIV-1 proteins are encoded by the 4-plasmid combination that composes the 3rd generation LVV transduction systems. LVVs use HIV-1-like mechanisms for viral genome integration and both transgene delivery and expression. LVVs rely on host cell machinery to transcribe and translate transgenes for either knocking down disease-causing genes and/or supplying functional genes in a targeted disease. LVVs integrate into host intronic and intergenic regions due to genomic accessibility, but there are no known biases toward specific target integration motifs. Investigation of LVV integration has uncovered the generation of chimeric LVV-host transcripts and altered host transcript splicing patterns. Several Food and Drug Administration (FDA)-approved LVV-derived therapies are used for treating diseases ranging from beta thalassemia to sickle cell anemia. An increasingly popular application of LVV is in the generation of chimeric antigen receptor (CAR) T cell therapies, which change and enhance T cell antigen specificity and effector function in liquid cancers. In November 2023, all CAR T cell therapies were placed under FDA investigation due to higher-than-expected rates of malignant transformation, hospitalization, and death in treated individuals. LVV integrations driving oncogene expression could be a cause for malignancy development. Current methods for resolving LVV integration patterns are technically limited by the sequencing approach applied allowing for only limited characterization of LVV integration profiles and altered host gene regulation. A comprehensive understanding of LVV integration and its consequences is necessary for understanding how these events influence host cell gene regulation and splicing, possibly identifying tunable variables for enhanced positive clinical outcomes. Here, we review the development of LVV systems, what is known about LVV integration patterns, technologies used to characterize patterns of LVV integration, and what is understood about the subsequent impact on host cell gene regulation and its potential linkage to patient malignancies.

ID: 41757835
Title: In vitro maturation 2.0: a new era for an underdog in ART.
Abstract: In vitro maturation (IVM) of human oocytes, once a pioneering concept predating conventional IVF, has long remained an underutilized technique in assisted reproduction. Despite early promise, clinical adoption of IVM has been limited due to lower embryo developmental competence and live birth rates compared to IVF. However, recent advances in the understanding of oocyte physiology, including cumulus-oocyte communication, the regulatory roles of cAMP/cGMP signaling pathways, and endocrine modulation of meiotic resumption, have reignited interest in optimizing IVM protocols. Innovations such as biphasic Capacitation (CAPA) IVM systems and the use of ovarian somatic support cells (OSCs) derived from induced pluripotent stem cells (iPSCs) aim to replicate the dynamic follicular environment more accurately and enhance oocyte competence. Clinical studies suggest that, while IVM still results in modestly lower cumulative live birth rates compared to conventional IVF, it offers significant advantages for selected patient populations, particularly women with polycystic ovary syndrome (PCOS), high ovarian reserve, or those requiring fertility preservation. Importantly, current evidence supports the genetic and epigenetic safety of IVM-derived offspring. As technical refinements continue and professional education expands, IVM is poised to fulfill its potential as a safer, less invasive, and more accessible alternative within the landscape of assisted reproductive technologies.

ID: 42327050
Title: Attenuated estrogen signaling disrupts placentation and drives trophoblast defects in Down syndrome.
Abstract: Proper establishment of the primitive placenta and subsequent tissue homeostasis in the mature placenta are critical for successful pregnancy in humans. Placental insufficiency is associated with adverse pregnancy outcomes, including fetal growth restriction, preeclampsia, and pregnancy loss. Moreover, emerging evidence suggests that placental defects are associated with long-term health challenges that manifest well into adulthood; yet the etiologies of such diseases are largely unknown. Defining the mechanistic basis for placental deficiencies, therefore, has important implications for improving both reproductive health and the lifelong well-being of affected children. Down syndrome is characterized by placental defects of unknown mechanistic origin, and notably, individuals with Down syndrome are at increased risk of developing diseases commonly associated with placental insufficiency later in life. Using induced pluripotent stem cells from Down syndrome patients, we found that stem cell-based embryo models (i.e., blastoids) and directed differentiation systems recapitulate trophoblast cell fate defects observed in placentas affected by Down syndrome. Furthermore, we demonstrate that attenuated estrogen signaling contributes to placental syncytialization defects and identify NRIP1, a transcriptional corepressor of estrogen receptor that is located on chromosome 21, as a key driver of trophoblast cell fate defects. Increased gene dosage of NRIP1 in an otherwise diploid cell line phenocopies cell fate defects observed in trophoblasts affected by Down syndrome. Our study suggests that estrogen signaling is a crucial regulator of trophoblast development and may serve as a potential target for therapeutic intervention. Estrogen signaling mediates syncytiotrophoblast fusionHuman iPS cells provide a tractable model for trophoblast defects in Down syndromeTrophoblast differentiation and estrogen signaling are disrupted in Down syndromeIncreased NRIP1 expression is sufficient to induce trophoblast defectsLogsdon and colleagues apply patient-derived induced pluripotent stem cells to recapitulate placentation defects observed in Down syndrome. The authors demonstrate that attenuated estrogen signaling disrupts trophoblast differentiation and identify NRIP1, a gene found on chromosome 21 that dampens estrogen signaling, as a regulator of trophoblast maturation. NRIP1 and estrogen signaling may represent important therapeutic targets for infertility and Down syndrome.

ID: 42369211
Title: Exploring the Functional Impact of Individual DDX41 Variants With a Fast and Robust Cell-Based Method.
Abstract: Germline DDX41 variants are found in up to 4% of patients with acute myeloid leukemia (AML) and myelodysplastic neoplasms (MDSs), with a higher prevalence among males and a late onset. In this study, we analyzed 647 Danish patients with suspected myeloid neoplasms to assess the frequency of germline DDX41 variants and the functional impact of variants of uncertain significance (VUSs) using a cell-based assay, CRISPR-Select. We identified 16 DDX41 variants in 30 patients, 14 of whom were confirmed or predicted as germline variants. Eight germline variants were classified as likely pathogenic/pathogenic (LP/P), and three as VUSs. We generated a monoallelic DDX41 K562 cell line and used it for CRISPR-Select assessment of variant effects on proliferation and survival. Pathogenic variants impaired proliferation, while benign variants did not. Missense variants had a more subtle effect than truncating ones, suggesting hypomorphic phenotypes. One DDX41 VUS had a significant negative effect on proliferation, suggesting it to be pathogenic, while the other two had no effect, suggesting them to be benign. These findings confirm a 2.4% frequency of LP/P DDX41 germline variants in Danish patients and demonstrate the value of CRISPR-Select in distinguishing pathogenic from benign variants.

ID: 42475285
Title: Protocol For Differentiating Pluripotent Stem Cells Into Primordial Germ Cell-like Cells.
Abstract: Using human pluripotent stem cell differentiation in vitro has significantly broadened the understanding of lineage specification events that occur during early development in vivo, a period that is extremely challenging to investigate. One of the first embryonic lineages to be specified in the human embryo is the germ cell lineage, marked by the induction of the primordial germ cells (PGCs). The PGCs are the sole founder population of the gametes later in life, and their specification is a critical first step towards the organism's fertility. Here, a robust protocol for generating PGC-like cells (PGCLCs) from human induced pluripotent stem cells (hiPSCs) is described, starting with the initial evaluation of the pluripotent state and cellular characteristics of the hiPSCs to ensure effective PGCLC differentiation. For this, the effects of different cell densities, plate coatings, and hiPSCs used for PGCLC differentiation are discussed. Finally, a workflow for characterization and quantification of hPGCLCs, using immunofluorescence and flow cytometry, is provided. The differentiation protocol focuses on conditions that are easy to establish and test in most laboratories and can be adjusted as necessary. Moreover, this protocol is fast, allowing determination of whether hiPSCs are suitable for PGCLC differentiation. The protocol aims to promote consistency and compatibility in hPGCLC outcomes across different hiPSC lines and culture platforms, ensuring robust yields that support reliable characterization and further optimization of downstream differentiation steps toward successful in vitro gametogenesis in humans.

ID: 42494498
Title: CRISPRing through time: How cutting-edge technology is revolutionizing life sciences and medicine.
Abstract: Given the plethora of emerging technologies, none have truly captured the minds as CRISPR. From the groundbreaking research, the ultimate battle of the prizes and patents to a number of books, the science of CRISPR continues to be significant in the biomedical field. For many decades now, the emergence of synthetic biology as an intervention to correct diseases has become the foundation of biomedical research. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based genetic editing has become a common place for routine investigation of scientific hypotheses in pre-clinical settings. More recently, CRISPR-based diagnostic testing kits for SARS-CoV-2 have showcased a translational output. Furthermore, a technological landmark was achieved when the Food and Drug Administration (FDA) approved the first CRISPR-based gene therapy (exa-cel) to edit erythroid specific enhancer region of BCL11A in hematopoietic stem cells, introduced in patients suffering from sickle cell anemia to achieve durable remission. In this review, we provide a snapshot into the most important milestones along the journey of CRISPR from its discovery in bacteria to its usage in precision medicine. The intervention of machine learning tools has now intertwined complex biology with high-throughput scalable outputs. Given the vast amount of information on CRISPR, we try to pin down key take-home messages for scientists as well as non-scientist readers. This review article attempts to understand why and how CRISPR remains significant and seamlessly integrates in the emerging era of new technologies.

ID: 42510769
Title: Oligonucleotide Synthesis Errors Are a Source of Untoward Variation in HDR-Mediated Gene Editing.
Abstract: Background/Objectives: Single-stranded oligonucleotides (ssODNs) are used as donor templates for therapeutic gene editing by CRISPR-Cas9 cleavage and homology-directed repair (HDR). Although ssODN sequence fidelity is critical to the safety and efficacy of editing, standard quality control methods cannot resolve individual nucleotide errors. Methods: We performed deep sequencing of ssODNs from three manufacturers and amplicons from edited hematopoietic stem/progenitor cells. Results: We find that synthesis errors are present in all ssODNs tested at rates that vary more than two-fold among manufacturers, at positions that are dependent on sequence context. These synthesis errors are propagated into the genome by HDR at frequencies proportional to their abundance in the ssODN. In our sickle cell mutation correction protocol, the most prevalent SNEs are predicted to produce benign β-globin variants, while the less frequent frameshift deletions are predicted to generate β-thalassemia-like alleles. Conclusions: Current quality control standards are insufficient to detect these errors, and deep sequencing of ssODNs should be incorporated into regulatory submissions for clinical gene editing programs.

ID: 42510922
Title: Gene-Edited Stem Cells for Ischemic Vascular Disease: Current Advances and Future Perspectives.
Abstract: Ischemic vascular diseases remain a leading cause of morbidity and mortality worldwide and are frequently associated with irreversible tissue damage. Although stem cell-based therapies have shown promise for vascular regeneration, their clinical translation has been limited by poor survival, insufficient engraftment, functional heterogeneity, and immune rejection. Recent advances in genome-editing technologies, including CRISPR/Cas9, base editing, and prime editing, have provided powerful tools for overcoming these limitations through precise genetic modification of stem cells. Gene editing can enhance angiogenic potential, improve resistance to ischemic stress, augment paracrine activity, promote endothelial maturation, and reduce immunogenicity. In this review, we outline the current genome-editing toolbox and its application to stem cell engineering for vascular regeneration in ischemic disease. We also examine emerging therapeutic concepts, universal donor cell platforms, and key issues in safety and ethics, with a focus on translational pathways. Taken together, advances at the interface of genome editing and stem cell biology are likely to accelerate the development of regenerative therapies that deliver more durable vascular repair in ischemic vascular disease.

ID: 42511925
Title: Gene Therapy for β-Haemoglobinopathies: From Molecular Correction to Curative Medicine.
Abstract: Background: β-haemoglobinopathies, including sickle cell disease and transfusion-dependent β-thalassaemia, are among the most common monogenic disorders worldwide and represent a major global health burden. Conventional treatments, such as blood transfusions, iron chelation, fetal haemoglobin induction, and allogeneic haematopoietic stem cell transplantation, have improved outcomes but remain limited by treatment-related toxicity, donor availability, and incomplete curative potential. Methods: A narrative literature review was conducted using PubMed up to 2025. Search terms included "sickle cell disease," "sickle cell anemia," "β-thalassemia," "transfusion-dependent beta-thalassemia," "gene therapy," "gene addition," "gene editing," "CRISPR-Cas9," "lentiviral vector," "children," "paediatric," and "pediatric." Relevant clinical trials, reviews, consensus statements, and guidelines were selected and qualitatively analysed. Results: Gene therapy for β-haemoglobinopathies is based mainly on two strategies: gene addition and gene editing. Gene addition uses lentiviral vectors to introduce functional or modified β-globin genes into autologous haematopoietic stem cells, whereas gene editing targets regulatory pathways, particularly BCL11A, to reactivate fetal haemoglobin synthesis or correct disease-causing mutations. Clinical studies have shown encouraging outcomes, including transfusion independence in many patients with β-thalassaemia and marked reduction or elimination of vaso-occlusive crises in sickle cell disease. Paediatric and adolescent data are increasingly promising, although still limited. Conclusions: Gene therapy is reshaping the treatment landscape of β-haemoglobinopathies by offering a personalised and potentially curative approach. However, long-term safety, conditioning toxicity, fertility preservation, accessibility, costs, and implementation in high-prevalence regions remain critical challenges. Further studies are needed to optimise patient selection and expand equitable access.

ID: 42518103
Title: Public knowledge, attitudes, and ethical views on CRISPR-Cas9 gene editing for genetic diseases in Taif, Saudi Arabia.
Abstract: Public support for gene editing, particularly for therapeutic purposes, remains strong. Recently, the Ministry of Health in Saudi Arabia approved CRISPR-Cas9 for treating Sickle Cell Disease and beta thalassemia. This study aims to assess the Taif population's opinion on gene editing and their knowledge of genetic modification. In this cross-sectional study, a questionnaire was distributed online from March 2, 2024, to June 15, 2024, to 747 residents of Taif City aged 18 and older. Among the respondents, 14.7% reported that they or their family members suffer from a hereditary disease, and 65.7% either work or study in the healthcare field or have a family member involved in healthcare. Additionally, 50.7% had previously heard of genetic modification. Marital status, number of children, and education level did not significantly influence opinions on genetic editing, whereas affiliation with the healthcare field was significantly associated with greater acceptance (p = 0.023), while a family history of hereditary disease showed a trend toward significance (p = 0.055). Public opinion strongly supports using genetic editing to treat life-threatening diseases in adults and embryos (63.2% and 73.6%, respectively). However, opinions are more divided on non-disease traits. Many respondents expressed interest in enhancing intelligence (73.8%) and strength (75.8%), as well as altering height (67.8%) and hair color (60.7%). While support was strong for therapeutic use, opinions were divided on enhancement, reflecting ethical tension despite high interest in modifying non-disease traits. Notably, 50.7% believed that using genetic editing for non-medical purposes crosses ethical boundaries and exceeds nature's limits. Awareness of gene-editing techniques was not significantly associated with acceptance (p = 0.108). In conclusion, public acceptance of gene editing in Taif is high, particularly among healthcare-affiliated individuals. Increasing public awareness remains essential to bridge ethical concerns and support informed engagement.

ID: 42528196
Title: Human iPSC-derived fetal granulosa-like cells enhance oocyte maturation outcomes and enable multi-generational safety evaluation in a mouse IVM model.
Abstract: In vitro maturation (IVM) of germinal vesicle oocytes may broaden assisted reproductive technologies; however, developmental competence remains limited by the lack of standardized follicular somatic support. We generated an endogenous FOXL2-P2A-tdTomato reporter human induced pluripotent stem cell (hiPSC) line using CRISPR/Cas9 knock-in and reporter-guided transcription factor (TF) programming to produce fetal granulosa-like cells (FGLCs). A flow cytometric TF screen identified TCF21, WT1-KTS, and NR1H4 as the strongest FOXL2 inducers, and combinatorial optimization showed that removing TOX3/ETV5 and adding NR2F2 substantially increased the FOXL2-positive fraction. Bulk RNA-seq, principal component analysis, and TF activity inference positioned the induced cells close to early-gestational human fetal granulosa cell profiles. In a mouse IVM, in vitro fertilization (IVF), blastocyst culture, and embryo transfer pipeline, FGLC supplementation did not change nuclear maturation rates but improved downstream development, most clearly increasing offspring production from cumulus-oocyte complexes. A comprehensive safety assessment of F0 offspring derived from FGLC-treated oocytes revealed a normal sex ratio, postnatal growth, gross anatomy, gonadal histology, and modified SHIRPA neurobehavioral profiles. Next-generation natural mating and germ cell assays demonstrated preserved fertility, normal IVF outcomes, and normal sperm parameters in the F1 generation. These results establish a scalable strategy for generating fetal granulosa-like supporting cells from hiPSCs and provide an effective and multi-generational safety framework for oocyte-contact IVM interventions.

ID: 42580028
Title: The future of clinical trials of mesenchymal stromal cells in acute graft-versus-host-disease.
Abstract: The U.S Food and Drug Administration approval of remestemcel-L for pediatric steroid-refractory acute graft-versus-host disease (GVHD) marks the culmination of 2 decades of efforts to translate mesenchymal stromal cells (MSCs). However, the path to this milestone reveals a pattern of MSCs failing in late-phase clinical trials. Multiple sources of heterogeneity may explain these results, including variability in donor and recipient biology, product manufacturing and characterization, and trial design. For example, certain subsets of acute GVHD patients, particularly those with inflammatory/immune activation, may derive the greatest clinical benefit from MSCs. Trials failing to stratify for this patient-level heterogeneity risk failure while obscuring signals of efficacy in responsive subgroups. Improving trial design considering heterogeneity requires leveraging advances in MSC manufacturing and refining clinical trial methodologies. The development of pooled and early-passage MSCs, induced pluripotent stem cell-derived (iPSC)-MSCs, biomaterial-encapsulated MSCs, and gene-edited or chimeric antigen receptor-expressing MSCs may reduce heterogeneity and improve trafficking to and survival at disease sites. Equally important, trial methodologies must explicitly address heterogeneity from MSC manufacturing and patient enrollment. Adaptive/Bayesian designs and master protocols may allow multiple biomarkers and MSC products to be tested simultaneously, with successful combinations being translated into practice, drawing from strategies that have been successful in precision medicine.

ID: 42584024
Title: 2026 Update on Clinical Trials in β-Thalassemia.
Abstract: The therapeutic landscape of β-thalassemia has evolved rapidly over the past decade, shifting from a historical reliance on transfusion support and iron chelation toward disease-modifying and potentially curative therapies. This review summarizes the clinical development of novel treatments for both non-transfusion-dependent and transfusion-dependent β-thalassemia, including approved therapies, agents in active development, and programs that have been discontinued. Disease-modifying strategies have focused on improving ineffective erythropoiesis, correcting iron dysregulation, and restoring red blood cell metabolism. Luspatercept and mitapivat have demonstrated clinically meaningful improvements in hemoglobin levels and reduction of transfusion burden and are now approved in multiple jurisdictions. Additional pyruvate kinase activators, such as etavopivat, are undergoing clinical evaluation. Curative approaches have advanced substantially through gene addition, gene editing, and base editing technologies. Approved therapies, including betibeglogene autotemcel and exagamglogene autotemcel, have achieved high rates of durable transfusion independence, while emerging platforms aim to further improve efficacy, safety, and accessibility. At the same time, several promising approaches targeting fetal hemoglobin induction, iron metabolism, and ineffective erythropoiesis have failed to demonstrate sufficient clinical benefit despite preclinical proof of concept, highlighting the complexity of therapeutic development in β-thalassemia. Future priorities include refining patient selection, generating real-world and comparative effectiveness data, developing clinically meaningful response criteria, expanding pediatric access, and addressing the substantial cost and accessibility challenges associated with novel therapies.

ID: 42587136
Title: Mechanistic machine learning for prediction of prime editing outcomes.
Abstract: Prime editing (PE) can make specific local changes to genomic DNA in living systems but its efficient application currently requires extensive optimization of PE guide RNA (pegRNA) sequences. Here we present OptiPrime, a machine learning model of PE efficiency based on current understanding of PE mechanisms. OptiPrime achieves state-of-the-art accuracy on PE efficiency prediction and enables prediction of nicking guide RNA (PE3) and dual pegRNA (twinPE) outcomes. We validate that OptiPrime has learned the determinants of mammalian mismatch repair (MMR) and is well suited for nominating MMR-evasive silent edits that improve PE efficiency. We demonstrate the use of OptiPrime in a variety of prospective therapeutic contexts in primary human and mouse cells. Lastly, we show that OptiPrime can be used to achieve streamlined and efficient in vivo correction of a pathogenic mutation in the brain of a mouse model of KIF1A-associated neurological disorder. We provide a webserver for OptiPrime ( https://optipri.me/ ) as a community resource.

ID: 42589126
Title: Cell-Based Therapies for Cardiac and Vascular Regeneration in Cardiovascular Disease: Recent Advances, Translational Barriers, and Future Directions.
Abstract: Cardiovascular diseases (CVDs) remain the foremost cause of death globally, responsible for 19.2 million deaths and 437 million disability-adjusted life years in 2023, with prevalent cases having more than doubled since 1990. No approved therapy restores myocardium lost to infarction. The adult heart replaces cardiomyocytes at approximately 1% per year in young adults, declining to about 0.45% per year with ageing, far below what is needed to recover the more than one billion cells destroyed by a large myocardial infarction. Cell-based regenerative strategies have been investigated for more than two decades, encompassing bone marrow mononuclear cells (BM-MNCs), mesenchymal stromal cells (MSCs), cardiac progenitor cells, cardiosphere-derived cells (CDCs), skeletal myoblasts, and induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). Safety has been consistent. Efficacy has been modest and variable: the CADUCEUS trial demonstrated scar mass reduction with CDCs without proportionate ejection fraction improvement; the Phase 1/2 POSEIDON trial confirmed MSC safety in 30 patients; and the Phase 3 DREAM-HF trial, enrolling 537 patients, failed its primary endpoint (HR 1.2, p = 0.406). Mechanistic work has established that transplanted cells engraft poorly and exert their benefit principally through paracrine signalling mediated by secreted extracellular vesicles and exosomes carrying microRNAs, trophic factors, and immunomodulatory proteins. For iPSC-CMs, electrophysiological immaturity and arrhythmogenic risk in primate models remain unresolved barriers. Emerging strategies include CRISPR-engineered hypoimmune iPSC lines, bioengineered cardiac patches, injectable hydrogel scaffolds, and engineered exosome platforms. This review provides a comprehensive synthesis of preclinical and clinical evidence, examines translational barriers, and identifies the scientific and regulatory priorities required before these therapies can enter routine clinical practice.

ID: 42589509
Title: Modeling Inherited Disorders of Post-Lanosterol Cholesterol Biosynthesis: From Animal Models to Patient-Derived Stem Cells.
Abstract: Altered post-lanosterol cholesterol biosynthesis causes a heterogeneous group of rare inherited metabolic disorders, including Smith-Lemli-Opitz syndrome, desmosterolosis, lathosterolosis, and congenital hemidysplasia with ichthyosiform nevus and limb defects syndrome. These conditions are characterized by impaired cholesterol synthesis together with the accumulation of disease-specific sterol intermediates. Current experimental and clinical evidence suggests that pathogenesis reflects both cholesterol insufficiency and sterol-mediated toxicity, including oxidative stress, perturbed developmental signaling, membrane dysfunction, and impaired neurodevelopment. Experimental models have played a central role in elucidating these mechanisms and in evaluating emerging therapeutic strategies. This review provides a comprehensive overview of currently available experimental models used to investigate inherited cholesterol biosynthesis disorders, including genetically engineered animal models, patient-derived fibroblasts, immortalized and CRISPR/Cas9-edited cell lines, and induced pluripotent stem cell-based systems. Particular emphasis is placed on Smith-Lemli-Opitz syndrome, the most extensively studied disorder within this group, while recent advances in modeling desmosterolosis, lathosterolosis, and congenital hemidysplasia with ichthyosiform nevus and limb defects syndrome are also critically discussed. We compare the strengths and limitations of each experimental platform, highlighting their contributions to understanding sterol metabolism, developmental abnormalities, and cell-type-specific disease mechanisms. Finally, we discuss current challenges and future perspectives, including the development of patient-specific induced pluripotent stem cell models, genome editing approaches, and next-generation multicellular systems. Collectively, this review provides an updated framework for selecting appropriate experimental models to investigate cholesterol biosynthesis disorders and accelerate the development of mechanism-based therapeutic strategies.

ID: 42589549
Title: Molecular Mechanisms Underlying Antimicrobial Resistance in Mycobacteria.
Abstract: Antimicrobial resistance in mycobacteria arises from a complex interplay of intrinsic and acquired mechanisms that collectively limit the efficacy of current therapeutic options. Intrinsic resistance is largely driven by the low permeability of the mycobacterial cell envelope, the activity of efflux pumps, and the presence of drug-modifying enzymes, which together restrict intracellular drug accumulation and contribute to broad baseline tolerance. This review integrates resistance mechanisms of both M. tuberculosis and M. abscessus, two clinically relevant mycobacteria that share core molecular pathways while exhibiting species-specific determinants that complicate treatment. Additional intrinsic factors, including biofilm formation and stress-induced adaptive responses, further enhance persistence and reduce susceptibility to multiple drug classes. Acquired resistance predominantly results from chromosomal mutations affecting drug targets or prodrug activation pathways, such as katG, inhA, rpoB, gyrA, and pncA in Mycobacterium tuberculosis, leading to high rates of multidrug-resistant and extensively drug-resistant disease. In nontuberculous mycobacteria, species-specific determinants-including inducible macrolide resistance mediated by erm(41) in M. abscessus, plasmid-mediated erm (55) variants, rrl and rrs mutations, diverse enzymatic inactivation systems, and regulatory alterations in the MarR family that result in inducible resistance to drugs such as ethionamide -generate highly variable resistance profiles that complicate treatment. Recent advances in molecular diagnostics, including PCR-based assays, whole-genome sequencing, CRISPR-based diagnostic platforms, AI-assisted diagnostics, and emerging multi-omics approaches, have improved the detection of resistance-associated mutations and enhanced understanding of mycobacterial pathophysiology. In parallel, new therapeutic agents and optimized regimens offer promising avenues to overcome resistance, although emerging resistance to novel drugs underscores the need for continued surveillance. This review synthesizes current knowledge on the molecular basis of resistance in M. tuberculosis and NTM, highlighting implications for diagnosis, treatment, and future research.

ID: 42589580
Title: Prime Editing Mediated Generation and Correction of the mdx5cv Mutation Restores Dystrophin Expression in Myoblasts.
Abstract: Duchenne muscular dystrophy (DMD) is caused by mutations in the DMD gene that abolish dystrophin expression. Prime editing enables precise genome modification without generating double-strand DNA breaks or requiring donor DNA templates. We established an in vitro prime editing workflow to generate and subsequently correct the mdx5cv mutation in mouse C2C12 myoblasts. Following optimization of engineered prime editing guide RNAs (epegRNAs) and PAM-flexible prime editors, wild-type cells were edited, clonally isolated, and genotyped. Mutation correction was then evaluated using optimized epegRNA designs. Two rounds of prime editing introduced the mdx5cv mutation into approximately 20% of alleles in C2C12 cells creating the mdx5cv C2C12 cell line. Clonal isolation yielded five homozygous mutant clones among 59 expanded colonies. Optimization studies identified an epegRNA containing a 16 nucleotide reverse transcription template and a 10 nucleotide primer binding site (RTT16/PBS10) as the most efficient design. Correction of the pathogenic allele reached approximately 26%, whereas longer PBS lengths reduced editing efficiency. In silico off-target analysis using Cas-OFFinder identified no candidate genomic loci with fewer than three mismatches for the spacer sequences used in either mutation generation or correction, suggesting a favorable predicted specificity profile. Corrected mdx5cv C2C12 myoblasts retained their capacity to differentiate into multinucleated myotubes. Representative Western blot analysis detected dystrophin protein expression in differentiated corrected mdx5cv myotubes, consistent with successful correction of the pathogenic mutation. These findings establish a robust prime editing platform for both the generation and correction of the mdx5cv mutation and provide proof of concept that precise correction of the pathogenic mutation is associated with restoration of dystrophin expression following myogenic differentiation.

ID: 42594274
Title: Histone H3K27M variants determine myeloid subset dependencies and immune reprogramming in diffuse midline glioma.
Abstract: Diffuse midline gliomas (DMGs) are highly aggressive, WHO grade 4 glial tumors that arise in midline central nervous system structures and are defined by K27M mutations in histone H3 genes. These K27M mutations shape intratumoral myeloid cell composition in DMG. In H3.1K27M DMGs, genetic ablation of monocyte recruitment reshapes the tumor microenvironment (TME) by reducing monocyte-derived macrophages (MDMs) and increasing microglia and neutrophil presence, with overall survival remaining unchanged, indicating compensatory myeloid remodeling is occurring. Here, by using CRISPR/Cas9-based genome editing, we generated a mouse model deficient for CCR1/CCR2/CCR3/CCR5 (Δ1235). Using this strain, we effectively abolished monocyte and MDM infiltration and reversed compensatory recruitment of CCR1+ neutrophils. Abolishing MDMs in tumors skewed remaining neutrophils and microglia toward a homeostatic state, reduced expression of immune checkpoint molecules on T cells, and extended the survival of H3.1K27M DMG-bearing mice. In contrast, H3.3K27M DMG showed independence from MDM recruitment, suggesting reliance on other TME-driven signaling. Last, H3.1K27M DMGs exhibited reduced microglia presence and a dose-dependent increase in MDM infiltration postirradiation. MDM depletion did not further enhance radiation efficacy, potentially due to compensatory recruitment of classical neutrophils. Collectively, these data reveal histone mutation-specific myeloid dependencies in DMG, highlighting MDM-independent mechanisms in H3.3K27M tumors and MDM-dependent pathways in H3.1K27M tumors.

ID: 42594811
Title: Pcgf5 controls the exit from totipotency in mouse embryonic stem cells.
Abstract: Mouse embryonic stem cell (ESC) cultures contain a rare subpopulation of two-cell-like cells (2CLCs) that transiently reactivate a two-cell embryo-like transcriptional program characteristic of zygotic genome activation (ZGA), including the endogenous retrovirus MERVL and Zscan4, and thereby regain a totipotent-like state. Polycomb repressive complex 1 (PRC1)-mediated H2AK119ub1 has been implicated in restraining entry into the 2C-like state through Pcgf6, yet the factors governing exit from this state and loss of totipotency remain poorly defined. Here, we show that among the six Pcgf paralogs, Pcgf5, which is most prominently upregulated in 2CLCs and forms an MERVL-driven chimeric transcript (Pcgf5MT2C_Mm) during ZGA in 2-cell embryos, controls exit from the 2C-like state in mouse ESCs. Using a reporter ESC line carrying MERVL-tdTomato and Zscan4c-EGFP (MtZG), we manipulated Pcgf5 dosage bidirectionally. Doxycycline (Dox)-inducible overexpression (OE) of Pcgf5 reduced the double-positive (DP) 2C-like population. Conversely, CRISPR-mediated knockout (KO) of Pcgf5 by targeting a common exon shared by all Pcgf5 variants (hereafter, total Pcgf5) increased the DP population. Time-lapse imaging directly confirmed that these changes reflected genuine differences in duration of the 2C-like state: OE shortened, whereas KO prolonged, the time cells spent in this state. These findings reveal that a Polycomb group factor controls not only entry into but also exit from the 2C-like state.

ID: 42595755
Title: Genome-wide CRISPR screening identifies cellular factors controlling nonviral genome editing efficiency.
Abstract: To systematically map cellular factors constraining nonviral genome editing, influencing uptake and intracellular trafficking, we develop a genome-wide CRISPR screening platform linking perturbation of 19,114 genes to editing outcomes in human cells. We identify six negative regulators of delivery whose depletion increases editing efficiency by up to six-fold across diverse payloads, loci, and cell types. We test the top two factors, GJB2 and BET1L, in two distinct human models: correction of a pathogenic adenine base mutation in KCNJ13 and introduction of a cytosine base mutation in the GABAA receptor gene. Depletion of either improves base-editing outcomes by 6-fold, potentially through effects on delivery. In a patient-derived model of retinal channelopathy, knockdown of either gene improves lipid nanoparticle base editing efficiency by over 3.5-fold. This enables functional restoration of Kir7.1 ion channels in a subset of edited cells, highlighting cellular barriers as actionable targets to enhance the potency of genetic therapies.

ID: 42597777
Title: Replicable generation and stable maintenance of rhesus macaque iPSCs for in vitro modeling of genetic frontotemporal dementia.
Abstract: At the Wisconsin National Primate Research Center, we have identified a family of rhesus carrying the microtubule-associated protein tau (MAPT) R406W mutation linked to frontotemporal dementia (FTD). Rhesus induced pluripotent stem cells (RhiPSCs) derived from these monkeys present a unique opportunity for in vitro modeling and comparison with cells derived from MAPT R406W human carriers. Here, we report the development of a reproducible method to generate RhiPSCs compliant with the standards of the International Society for Stem Cell Research (ISSCR) to support in vitro modeling of FTD-MAPT R406W. Our stepwise approach identified efficient methods for fibroblast derivation, fibroblast reprogramming to RhiPSC, and RhiPSC maintenance over continued culture. To derive fibroblasts from MAPT wild type (WT) and R406W monkeys, a combination of manual processing and overnight enzymatic digestion was required to maximize the number of low passage fibroblasts available for reprogramming. Fibroblast reprogramming to RhiPSC using Sendai viral vectors versus oriP/EBNA1 episomal plasmids revealed the latter as most efficient. Electroporation conditions for oriP/EBNA1 reprogramming were optimized to maximize plasmid uptake and cell survival. Ultimately, eight RhiPSC lines were derived from 4 donor rhesus monkeys (n = 2 WT, n = 2 R406W; two clonal lines per donor) and fully characterized according to ISSCR standards. RhiPSC stemness and genetic stability was best maintained on mouse embryonic fibroblast feeders in Universal Primate Pluripotency Stem Cell medium, as opposed to Essential 12 medium supplemented with IWR1, which produced cytogenetic abnormalities. Rhesus neural progenitor cells were generated using a monolayer protocol and expressed PAX6 and NESTIN after 21 days of differentiation. Our reliable method will be useful to labs seeking to derive RhiPSCs for preclinical studies. Overall, the RhiPSCs generated from MAPT R406W carriers will be a critical resource for evaluating the molecular underpinnings of tau-related neurodegeneration across primate species.

ID: 42599088
Title: Liver Organoids: From Disease Modelling to Regenerative Medicine.
Abstract: Liver organoids are three-dimensional miniature liver models that recapitulate the complex architecture and key functions of the human liver in vitro, offering powerful platforms for both fundamental research and translational applications. This review systematically summarises current fabrication strategies, disease-modelling utilities and regenerative potentials of liver organoids, alongside the major challenges and future directions. In recent years, the field has witnessed several breakthroughs. Through endothelial co-culture approaches, vascularised and metabolically zonated liver organoids have been successfully generated, achieving endothelial coverage exceeding 85%. Prime editing enables precise correction of pathogenic mutations in patient-derived organoids, with no off-target effects detected at the genome-wide level. In disease modelling, iPSC-derived liver organoids faithfully recapitulate the pathological progression of metabolic dysfunction-associated steatotic liver disease (MASLD) and verify the lipid-lowering efficacy of semaglutide. Macrophage-integrated organoid models support the full life cycles of HEV, SARS-CoV-2 and dengue virus, providing new tools for antiviral drug screening. Large-scale patient-derived tumour organoid biobanks successfully preserve the heterogeneity and clinical drug-resistance signatures of liver cancers. In regenerative medicine, encapsulated hepatocyte organoids and the UTOpiA bioartificial liver system have effectively rescued acute liver failure in animal models, while gene-edited autologous organoids offer potential curative strategies for genetic disorders such as Wilson disease. Nevertheless, insufficient hepatocyte functional maturity, difficulties in constructing vascular networks, and the lack of standardised culture protocols remain major obstacles to clinical translation. By bridging fundamental liver biology and clinical practice, liver organoid technology lays a solid foundation for precision hepatology and regenerative therapies. Continued interdisciplinary efforts are still required to overcome current limitations and facilitate its clinical adoption.

ID: 42599816
Title: CRISPR RNA Engineering Enables Single Nucleotide Polymorphism Discrimination in Nucleic Acid Detection.
Abstract: Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based diagnostic systems have emerged as powerful platforms for sensitive nucleic acid detection, yet their clinical reliability is frequently constrained by off-target activation and insufficient discrimination of closely related sequences. This review synthesizes current advances aimed at enhancing the specificity of CRISPR diagnostics, with particular emphasis on the pivotal role of CRISPR RNA (crRNA) engineering. We detail how structural determinants of crRNA, including spacer length optimization, intentional mismatch design, secondary-structure modulation, chemical modification, strand-displacement gating, and synergistic design frameworks, govern CRISPR-mediated target recognition and define the energetic and kinetic thresholds for accurate cleavage. Key engineering strategies encompassing computational prediction and modeling, high-throughput screening, and hybrid guide architectures are systematically examined for their capacity to elevate single-nucleotide discrimination, stabilize reaction performance, and enable robust multiplexed detection for pathogen profiling and mutation identification. Despite rapid progress, outstanding challenges persist, including interference from complex clinical matrices, lack of unified evaluation standards, and scalability barriers that hinder clinical translation. Addressing these limitations through integrated crRNA design, system-level optimization, and standardized benchmarking will be essential for realizing the promise of CRISPR diagnostics. Ultimately, these advances are poised to support ultrasensitive, highly specific, and portable point-of-care testing, thereby accelerating precision medicine and strengthening infectious disease surveillance and management.

ID: 42600889
Title: Unlocking the Curative Potential of Gene Transfer and Editing for Hematopoietic Disorders.
Abstract: Hematopoietic stem and progenitor cells (HSPCs) gene therapy may transform the therapeutic landscape for inherited hematological disorders and already offers potentially curative options for some diseases, including inborn errors of immunity and β-hemoglobinopathies. Its continued success relies on further refinement of gene transfer technologies, gene editing tools such as CRISPR-Cas, and optimized ex vivo HSPC manipulation protocols that ensure robust, long-term engraftment and clonal diversity with reduced-toxicity, non-genotoxic conditioning strategies. Here, we review recent developments and refinements in gene transfer and editing technologies for HSPCs, while also discussing the critical limitations and hurdles to clinical translation, as recently presented at the New Investigator Committee Gene Therapy webinar. Future directions must prioritize integrating technological innovation with the development of equitable and simplified models to reduce costs and ensure that these life-saving cellular therapies reach patients worldwide. Teaser abstract Hematopoietic stem and progenitor cells (HSPCs) gene therapy is advancing rapidly, with lentiviral gene transfer, genome editing, and emerging in vivo delivery approaches expanding the therapeutic landscape for inherited hematologic disorders. Recent clinical successes have demonstrated the potential for durable correction, while ongoing refinements continue to improve safety, efficacy, and feasibility. Key challenges remain in genotoxicity, conditioning toxicity, manufacturing scalability, and equitable global access.

ID: 42600901
Title: The Bombyx mori ortholog of protein-cysteine N-palmitoyltransferase (Rasp) is essential for the development of adult organs.
Abstract: The gap (apterous and rudimentary gonads) mutation is a spontaneous mutation that causes defects in adult organs in the silkworm Bombyx mori. Although gap larvae exhibit normal external morphology, gap mutants exhibit severe defects in adult organs, suggesting that the gap gene is required for the development of imaginal discs and the primordia of adult organs. Genetic analysis of the gap mutation provides an opportunity to understand the molecular mechanisms underlying the development of adult organs. In this study, we performed RNA-seq analysis of ovaries from gap mutants. De novo assembly of RNA-seq reads identified the insertion of a retrotransposon in the gene encoding the Bombyx ortholog of protein-cysteine N-palmitoyltransferase (Rasp), which is required for Hedgehog signaling in Drosophila. PCR and subsequent sequencing analyses revealed that an LTR transposon is inserted into exon 1 of the BmRasp gene. To verify the involvement of BmRasp in the gap phenotype, we generated a knockout allele of BmRasp (BmRaspKO) using the CRISPR/Cas9 system. Both homozygous knockout mutants and F1 pupae with the gap/BmRaspKO genotype exhibited gap-like phenotypes characterized by apterous pupa, indicating that BmRasp corresponds to the gap locus. To investigate why zygotic BmRasp expression is dispensable during embryogenesis but essential for the development of adult organs, we analyzed BmRasp expression levels in eggs and larval tissues. Furthermore, testis transplantation experiments demonstrated that BmRasp is dispensable for spermatogenesis, despite the fact that the testis exhibits the highest expression level of BmRasp among the tissues analyzed. Finally, we discuss the physiological roles of Hedgehog signaling in embryogenesis and in the development of imaginal discs in B. mori.

ID: 42602967
Title: TP53 mutation drives unique transcriptional and functional vulnerabilities independent of del(17p) in multiple myeloma.
Abstract: TP53 abnormalities contribute to treatment resistance and poor prognosis in multiple myeloma (MM), yet their functional consequences remain unclear. Here, we integrate ex vivo drug sensitivity profiling, genomics, transcriptomics, and proteomics across 167 CD138+ bone marrow patient samples to characterize TP53-associated vulnerabilities. Genome-wide CRISPR-Cas9 and RNAi screening identify vulnerabilities in TP53-mutated MM, with or without del(17p), highlighting the dependency on spindle organization, mitotic regulation, DNA synthesis, and transcriptional and metabolic regulation, but independence from MDM2. CD138+ cells with TP53 mutation exhibit increased sensitivity to chemotherapeutics, HDAC, HSP90, IGF1R, and PI3K/AKT/mTOR inhibitors as well as RNA synthesis inhibitor plicamycin, with distinct drug response profiles of MM with del(17p) and WT TP53. Our study provides new insights into refining TP53 classification to optimize treatment strategies for high-risk MM.

ID: 42603820
Title: Neurabin I haploinsufficiency disrupts ion channel regulation and synaptic maturation in human cortical neurons in neurodevelopmental disorders.
Abstract: Heterozygous loss-of-function variants in Neurabin I (PPP1R9A), responsible for encoding a cytoskeletal scaffolding protein essential for synaptic plasticity, are recurrently associated with neurodevelopmental and neuropsychiatric disorders, yet their direct effects on human neuronal maturation remain unclear. Here, we establish the first comprehensive human mechanistic model of PPP1R9A haploinsufficiency using an isogenic CRISPR/Cas9-engineered iPSC system differentiated into cortical neurons to define dosage-dependent functional consequences. PPP1R9A+/- neurons exhibited pronounced hyperspinogenesis and increased neuritic complexity, indicative of aberrant structural maturation; however, whole-cell patch-clamp recordings revealed impaired intrinsic excitability, including reduced action potential firing, altered waveform properties, and defective axo-somatic coupling, uncovering a striking dissociation between neuronal morphology and function. Long-read single-cell transcriptomics and quantitative proteomics identified coordinated downregulation of ion channel and synaptic transmission pathways, including genes essential for sodium channel function and glutamatergic signaling, together with disruption of synaptic vesicle cycling, axon guidance, and neurodevelopmental programs. Pseudotime trajectory analysis further demonstrated delayed neuronal differentiation, with mutant neurons accumulating at intermediate developmental states rather than acquiring mature cortical identities. Importantly, molecular rescue experiments confirmed causality, as restoration of full-length PPP1R9A expression robustly normalized transcriptional and synaptic signaling programs, whereas allele-specific antisense oligonucleotide-mediated suppression of the mutant transcript achieved only partial rescue. Taken together, these findings establish PPP1R9A haploinsufficiency as a driver of impaired molecular, electrophysiological, and developmental maturation in human cortical neurons, providing a human-specific mechanistic framework linking reduced Neurabin I dosage to neurodevelopmental and psychiatric disease risk.

ID: 42606179
Title: Repeated mutation of a GT92 glycosyltransferase gene confers antiviral resistance in two Caenorhabditis species.
Abstract: Host-pathogen interactions evolve rapidly within species, providing natural genetic resources for the identification of specific ecological interaction factors. We previously identified RNA viruses that infect the nematodes C. elegans and C. briggsae in a species-specific manner. Wild strains of both host species demonstrate ample variation in viral sensitivity. Here we use recombinant inbred lines and pool-sequencing approaches to genetically map a major resistance locus in the C. elegans MY10 strain, narrowing down its position by CRISPR/Cas9 mediated recombination and testing candidates by genome editing. A rare non-synonymous polymorphism in the gtnt-1 gene, encoding a putative glycosyltransferase of the GT92 family, causes resistance to viral infection in MY10. Loss-of-function gtnt-1 alleles conferred host protection only at late developmental stages, highlighting the importance of multigenerational assays capturing the full course of viral infection and transmission. Viral resistance through gtnt-1 mutation occurred repeatedly in C. elegans, with diverse alleles each remaining at low frequency (<1%). Furthermore, leveraging closely related C. briggsae strains differing in viral susceptibility, we find that repeated loss-of-function alleles of the Cbr-gtnt-1 ortholog similarly enhanced resistance and host fitness upon infection. In conclusion, recurrent evolution in two host species of loss-of-function alleles of gtnt-1 orthologs leads to viral resistance. The gtnt-1 gene being conserved between Caenorhabditis species, these repeated inactivation events provide a case of transient ecological adaptation to a pathogen through recurrent mutation of the same gene in two species. The low population frequencies of resistant alleles point to a changing eco-evolutionary context that prevents their spread in populations.

ID: 42607937
Title: Integration of PNA-mediated PCR and CRISPR/Cas13a for highly sensitive detection of EGFR T790M mutation in circulating tumor DNA.
Abstract: Circulating tumor DNA (ctDNA) is characterized by low abundance and fragmentation, limiting the development of genetic variant detection technologies. In this study, we established a highly sensitive and specific assay by combining peptide nucleic acid (PNA)-mediated PCR clamping with CRISPR/Cas13a trans-cleavage detection. A PNA probe targeting the wild-type (WT) EGFR T790M allele was designed to suppress WT amplification during PCR, while minimally affecting mutant allele amplification. By combining the target specificity of Cas13a for mutant alleles with the WT-suppression capability of PNA-PCR, we achieved a dual-enrichment effect for mutant detection. When applied to EGFR T790M mutation detection, the assay reached an analytical sensitivity of 0.02%. We established a standard curve for T790M detection using cell-free DNA standards. Clinical validation in 20 plasma samples from lung adenocarcinoma patients demonstrated that the PNA-Cas13a assay achieved a diagnostic sensitivity of 93.3% (95% CI: 68.1%-99.8%) and a specificity of 100% (95% CI: 47.8%-100%), with detection concordance comparable to or improved over ARMS-PCR in this pilot cohort. The results suggest its preliminary diagnostic utility in liquid biopsy. In conclusion, the PNA-Cas13a assay enables sensitive and specific detection of EGFR T790M mutations in ctDNA, is readily adaptable to multiple gene loci, and holds promise for clinical monitoring of tumor drug resistance.

ID: 42607939
Title: The miRNA-virus axis in RNA virus infections: Mechanisms, therapeutic targeting, and systems biology approaches.
Abstract: RNA viruses are a diverse and rapidly evolving group of pathogens that significantly contribute to worldwide morbidity and mortality, propelled by elevated mutation rates and effective use of host cellular mechanisms. MicroRNAs (miRNAs) have emerged as an essential post-transcriptional regulator of host-virus interactions, influencing viral replication, immunological responses and disease progression. This review offers a comprehensive overview of the miRNA-virus axis in RNA virus infections, highlighting the dynamic and context-dependent roles of host miRNAs. We investigate the mechanistic basis of miRNA-mediated regulation, including direct targeting of viral RNA, modulation of host dependency factors, regulation of antiviral signaling pathways, and immune-mediated effects on infection outcomes. We propose a functional categorization of miRNAs that transcends the conventional antiviral-proviral dichotomy, facilitating a more accurate comprehension of their involvement in various infection settings. This review connects mechanistic insights with translational applications by outlining novel treatment techniques, such as miRNA mimics, anti-miRs, RNA interference, and CRISPR-based methodologies. We also propose a therapeutic decision framework that links miRNA functional classification with targeted antiviral interventions. Key challenges including delivery efficiency, tissue specificity, and off-target effects are also discussed, with emphasis on lipid nanoparticles and exosome-based delivery systems. We further highlight the role of systems biology approaches, including multi-omics integration, single-cell and spatial transcriptomics, network analysis, and artificial intelligence, in identifying regulatory miRNA hubs and enabling precision antiviral medicine. Collectively, this review reinterprets the miRNA-virus axis as a dynamic regulatory network and provides a translational framework for the development of next-generation antiviral therapeutics.

ID: 42608059
Title: The combination of morphogenic regulators BABY BOOM and GRF-GIF improves maize transformation efficiency and promotes leaf regeneration.
Abstract: Transformation is an indispensable tool for plant genetics and functional genomics. Although stable transformation in maize is no longer a major obstacle, there remains a need for accessible and efficient methods for academic laboratories. Here, we present the GGB system, a rapid and efficient approach optimized for immature embryo transformation in B104 and other maize lines. This system combines two distinct morphogenetic regulators, the wheat GRF4-GIF1 chimera and the maize BABY BOOM (BBM) transcription factor (hence the name "GGB") with a modified QuickCorn protocol, enabling regeneration of transformed maize plantlets in c. 2 months with an efficiency 7-fold higher than when compared to either morphogenic factor used in isolation. Expression of both regulators did not significantly affect development, eliminating the need to excise them after regeneration. However, transmission of the transgenic GGB construct through pollen was significantly reduced, potentially aiding transgenic line containment. We show that the GGB system is adaptable for CRISPR-Cas9 editing and reporter line generation. Furthermore, stable GGB transformants exhibited high leaf regeneration capacity via somatic embryogenesis. RNA-seq time-course profiling of GGB leaf cultures identified additional factors that could promote regeneration and led to the discovery of asparagine and trehalose as additional media components that significantly enhanced leaf regeneration.

ID: 42610637
Title: Assembly and Maintenance of Myofibrils in the Heart.
Abstract: The assembly of actin and myosin to the paracrystalline arrangement of myofibrils in striated muscle, that is, heart and skeletal, has puzzled many a cell biologist. Different models, based on cultured cardiomyocytes, were proposed for the last 30 years to explain the sequence of events. These were then evaluated during heart development in embryos in situ and more recently in cardiomyocytes that were derived from human induced pluripotent stem cells (iPSC-CMs). A lot of the initial work was quite descriptive but recent advances in gene editing technology and in super resolution microscopy allow a more targeted functional analysis and will give unprecedented insight. This review will discuss the general principles of myofibril assembly in the heart and cover new findings on myofibril maintenance and turnover.

ID: 42610742
Title: Energetic Determinants of Expanded PAM Recognition in Engineered FnCas9.
Abstract: Expanding protospacer-adjacent motif (PAM) compatibility while preserving specificity remains a central challenge in CRISPR-Cas9 engineering. Francisella novicida Cas9 (FnCas9) exhibits high intrinsic specificity but is constrained by stringent PAM requirements. Here, we quantitatively examine the energetic and catalytic consequences of PAM-interacting mutations in three engineered variants, en1 (E1369R), en15 (E1603H), and en31 (G1243T/E1369R/E1449H), using a VEGFA3 DNA substrate framework. Microscale thermophoresis and isothermal titration calorimetry reveal that the engineered variants enhance binding affinity toward the canonical NGG PAM relative to wild-type FnCas9, with modest gains in binding free energy. Selected noncanonical PAM substrates, particularly TGA and TAG, also show improved binding by en15 and en31, with en31 displaying the strongest overall binding among the substrates tested. Thermodynamic profiles indicate that enhanced affinity is associated with more favorable enthalpic contributions, consistent with altered interactions at the PAM interface; however, the specific molecular contributions underlying these changes remain to be directly established. Despite improved binding, active-site titration reveals reduced fractions of catalytically competent enzyme in engineered variants, particularly en31, necessitating higher enzyme concentrations to achieve cleavage efficiencies comparable to wild-type. Cleavage assays demonstrate that en31 most effectively couples improved recognition of the tested noncanonical PAM substrates to productive catalysis, enabling robust cleavage of both TGA and TAG substrates while maintaining minimal off-target activity under the conditions examined. Together, these results suggest that PAM-interacting mutations in FnCas9 can alter the energetic coupling between DNA binding and catalytic activation within the VEGFA3 substrate framework tested, highlighting the importance of balancing substrate affinity with conformational activation in the design of high-precision genome-editing nucleases.

ID: 42611132
Title: A novel needleless delivery of Cas9 ribonucleoprotein complex in Bemisia tabaci embryos for gene editing.
Abstract: The CRISPR/Cas9 system, particularly the Cas9-sgRNA ribonucleoprotein (RNP) complex, offers a highly efficient platform for gene editing. However, traditional microinjection methods for RNP delivery in insect embryos are labor-intensive, technically demanding, and often reduce embryo viability, especially in species with fragile, microscopic embryos. In this study, a novel, non-invasive delivery method for the RNP complex in tiny insect embryos has been optimized. Whitefly, Bemisia tabaci, an invasive insect pest of agricultural importance and vector of plant diseases, was considered as a model organism. A needleless dechorionation-permeabilization method was employed to deliver the RNP complex to the early embryonic stage of B. tabaci embryos. B. tabaci heat shock protein 70 (hsp70) gene, which interacts with the begomovirus coat protein, aiding in its internalization and successful transmission by B. tabaci in a persistent circulative manner, was targeted for knockout. Two sgRNAs were synthesized via in vitro transcription, and the Cas9-sgRNA complexes were validated by in vitro cleavage assays. The localization of the GFP-labelled Cas9-sgRNA complex confirmed successful RNP delivery, as observed through confocal microscopy. A survival rate of 18% of the embryos was recorded post-RNP delivery. Sequencing of treated embryos showed 25- and 28-nucleotide deletions in the hsp70 exon. Synthego ICE analysis revealed up to 84% gene knockout efficiency. This method enables batch processing of embryos, drastically reducing delivery time and associated costs while improving throughput. Hsp70 KO B. tabaci mutants generated in the study are expected to be incompetent begomovirus transmitters, which would help restrict the spread of the virus. Our study overcomes a key bottleneck in CRISPR/Cas delivery to small insect embryos, opening new avenues for rapid, high-throughput, and cost-effective RNP delivery methods in insect embryos. The novel non-invasive methods would be helpful in the deployment of gene editing for sustainable pest control.

ID: 42611583
Title: CRISPR/Cas9-Mediated Site-Directed Mutagenesis of Genes in Klebsiella pneumoniae.
Abstract: Constructing target-gene mutants with a common genetic background is crucial for elucidating gene function in antimicrobial resistance (AMR) research. Taking advantage of the single-guide RNA (sgRNA) and protospacer adjacent motif (PAM) sequence (3'-NGG) specificity of the Cas9 protein in the CRISPR/Cas9 (Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) system and codon degeneracy, the authors design a repair template that incorporates the desired point mutation while excluding the PAM sequence disrupted by a synonymous substitution, thereby preventing re-cleavage by CRISPR/Cas9. This method employs two available plasmids, pCasKP-apr and pSGKP-spe, offering straightforward operation and high screening specificity. As a result, the approach enables efficient generation of genetically defined mutant strains of Klebsiella pneumoniae (K. pneumoniae) and is readily adaptable to routine laboratory settings. Furthermore, the protocol minimizes off-target editing, shortens experimental timelines, reduces screening workload, and provides a reliable platform for investigating resistance mechanisms, validating candidate genes, and supporting functional genomics studies in clinically relevant bacterial pathogens.

ID: 42611607
Title: Light- and X-ray-Activated Liposomes for Controlled Gene Editing and Drug Delivery.
Abstract: Photodynamic therapy (PDT) exploits photosensitizer activation to generate reactive oxygen species (ROS), principally singlet oxygen. Beyond direct cytotoxicity, this photochemistry can be repurposed for on-demand cargo release from lipid nanocarrier systems, enabling spatiotemporal control over therapeutic delivery that is not achievable with conventional lipid nanoparticles. This protocol presents methods for fabricating and characterising two distinct verteporfin (VP)-integrated lipid nanoparticle formulations: (1) light-triggered liposomes composed of DOTAP, DOPE, cholesterol, and VP for delivery of CRISPR-Cas9 ribonucleoprotein (RNP) complexes; and (2) X-ray-triggered liposomes composed of DOTAP, DOPC, VP, and gold nanoparticles for controlled chemotherapy drug release. Upon activation at 690 nm (visible light) or by clinical X-ray radiation (6 MeV), VP generates singlet oxygen that oxidises unsaturated lipid components, destabilising the nanoparticle membrane and releasing encapsulated cargos. Protocols are provided for liposome formulation by thin-film hydration and membrane extrusion, physicochemical characterisation, light- and X-ray-triggered cargo release assessment, in vitro gene knockout in human cells, and in vivo validation using a quantitative zebrafish visual reporter system and a mouse xenograft tumour model. Representative results demonstrate knockout of up to approximately 326 slow-muscle fibres per zebrafish embryo via light activation and significant tumour growth suppression via X-ray-triggered doxorubicin release.The clinical precedent for 689-690 nm verteporfin activation in the eye motivates evaluation of this platform for ophthalmic delivery, although retinal biodistribution, pharmacokinetics, and large-animal safety remain to be established. These methods provide an experimental approach with translational potential for externally controlled therapeutic cargo release.

ID: 42612101
Title: Generation of GABAergic Neurons from Human Induced Pluripotent Stem Cells Using Doxycycline-Inducible ASCL1/DLX2 Expression at AAVS1.
Abstract: Human induced pluripotent stem cells (hiPSCs) offer an unprecedented opportunity to model human neurological diseases in vitro. Reliable generation of defined neuronal populations, such as gamma-aminobutyric acid (GABAergic) neurons, is essential for these studies. This protocol details a robust method for differentiating hiPSCs into GABAergic neurons using a doxycycline-inducible system. The approach relies on the stable integration of transgenes encoding the transcription factors ASCL1 and DLX2 into the safe-harbor adeno-associated virus integration site 1 (AAVS1.) locus via CRISPR/Cas9-mediated knock-in. This targeted integration avoids the transgene silencing often observed with random lentiviral integration. The protocol covers the maintenance of hiPSCs, the nucleofection process for transgene integration, puromycin selection to enrich successfully engineered cells, and the step-by-step differentiation procedure. Upon induction with doxycycline, the engineered hiPSCs rapidly exit the cell cycle, acquire neuronal morphology, and express GABAergic lineage markers within 21 days. Key steps include neuro-induction and subsequent maturation using specific growth factors, as well as potential approaches to improve cellular adhesion during maturation. This standardized method yields enriched populations of induced neurons expressing GABAergic lineage markers, providing a valuable tool for neuroscience research and cellular modeling.

ID: 42612103
Title: Genome Editing in Primary Mammalian Cells via Electroporation of Editor RNA.
Abstract: CRISPR editors including nucleases, base editors, and prime editors can efficiently correct disease-causing genetic variants or disrupt target genes. Editing outcomes are commonly evaluated in cultured primary cells, patient-derived cells, or engineered cell lines to study the impact of genetic variation or as a first step before initiating animal studies or clinical translation. Delivery of editors as mRNA together with synthetic guide RNAs into mammalian cells can improve editing efficiency relative to plasmid-based approaches and prevent issues such as DNA integration or off-target editing from sustained expression. This article presents a workflow to prepare genome editor mRNA by in vitro transcription (IVT), including co-transcriptional capping and chemically-modified nucleotides, electroporate editor mRNA and guide RNAs into primary human fibroblasts, induced pluripotent stem cells (iPSCs), or lymphoblastoid cell lines (LCLs), and quantify editing outcomes by targeted amplicon sequencing on an Illumina platform followed by analysis using CRISPResso2. This workflow enables quantitative benchmarking of guide RNAs, electroporation parameters, and editor variants, and supports downstream applications including single-cell cloning, phenotypic assays, preclinical animal studies, and therapeutic development.

ID: 42612243
Title: CRISPR-Cas9-induced genetic mosaicism in three species of the microcrustacean Daphnia.
Abstract: Genetic mosaicism can arise from in vivo CRISPR-Cas9 gene editing, especially in the embryos. This study evaluates the extent of genetic mosaicism resulted from CRISPR-Cas9-mediated knockout for 11 genes in the freshwater microcrustacean Daphnia magna, D. pulex, and D. sinensis. Based on extensive genotyping data of the asexually produced progenies of successfully edited females, we find strong evidence of mosaicism in 9 of these genes. The genotyping data also suggest the gene editing activity can take place as early as the one-cell embryo stage and extends into the 32-cell and later stages. This study establishes genetic mosaicism as an important feature of Cas9-mediated gene editing in Daphnia.

ID: 42612424
Title: Development of a human iPSC and patient phenotyping resource for preclinical investigations of neurodevelopmental disorders.
Abstract: In this manuscript, we report the development of a comprehensive resource designed to harness the transformative potential of patient-derived induced pluripotent stem cells (iPSCs) to advance the study of neurodevelopmental disorders (NDDs). Using CRISPR-Cas-mediated genome editing, the Human Neuron Core generated a repository comprising 29 isogenic iPSC pairs, two sex-matched parental control iPSC pairs, and one unmatched patient line representing six monogenic NDDs: Tuberous Sclerosis Complex, PTEN Hamartoma Tumor Syndrome, KCNQ2 Developmental and Epileptic Encephalopathy, FOXG1 Syndrome, Phelan-McDermid Syndrome, and SETBP1 Haploinsufficiency Disorder. In parallel, detailed clinical phenotyping data were collected to enable comparison of cellular phenotypes with clinical severity in future studies. This integrated collection of genetically defined iPSC lines and associated clinical data provides a powerful platform for investigating disease mechanisms and advancing iPSC-based drug discovery for NDDs.

ID: 42617141
Title: Translating CRISPR to Practice in the Clinic: Transformative Therapy in Hemoglobinopathies and Emerging Applications in Malignancies.
Abstract: Exagamglogene autotemcel, the first clustered regularly interspaced short palindromic repeats (CRISPR)-based gene-editing therapy to enter clinical practice, has established genome editing as a curative-intent option for eligible patients with severe sickle cell disease (SCD) and transfusion-dependent β-thalassemia (TDT). Ex vivo CRISPR-Cas9 editing of autologous CD34-positive hematopoietic stem and progenitor cells to reactivate fetal hemoglobin has produced durable freedom from severe vaso-occlusive crises in SCD and sustained transfusion independence in TDT. This approach offers a donor-independent alternative to allogeneic hematopoietic stem-cell transplantation without graft rejection or graft-versus-host disease. Hemoglobinopathies, therefore, provide the first clinically validated delivery model for CRISPR therapeutics. Successful implementation, however, requires more than editing efficacy. It depends on structured referral, candidacy assessment, organ function review, mobilization and collection, centralized manufacturing, pharmacokinetic-guided myeloablative conditioning, transplant-level supportive care, fertility preservation, psychosocial support, and prolonged surveillance coordinated among primary hematologists and cellular therapy programs. Key barriers include stem cell collection, conditioning-related toxicity, cost, reimbursement friction, and persistent inequities in access. Long-term follow-up and registry participation are necessary to evaluate outcomes beyond pain crises, identify late toxic effects, and compare real-world effectiveness across gene editing, gene addition, and allogeneic transplantation. In oncology, CRISPR-based therapy remains investigational, with early clinical feasibility demonstrated in relapsed or refractory B-cell malignancies, T-cell malignancies, AML, multiple myeloma, and selected solid tumors. For hematologists, oncologists, and transplant and cellular therapy programs, the central challenge is to integrate CRISPR into practice with the rigor required for any high-risk curative therapy: careful patient selection, disciplined delivery, and long-term accountability.

ID: 42620346
Title: APP Dosage and Extracellular Domain Variants Drive Distinct Defects in Neurogenesis modeled in Down Syndrome iPS cells.
Abstract: The amyloid precursor protein (APP) is heavily studied as the source of amyloid beta in Alzheimer's disease (AD), however, the complex functions of APP remain poorly understood, as does the impact of APP dosage on neurodevelopment. Here we study APP specifically in the context of Trisomy 21. In an effort to reduce APP dosage in trisomy 21 iPSCs, we generated trisomic isogenic lines which vary in APP dosage, including a full APP knock-out line, as well as lines carrying mutations of the APP extracellular domain. We used a panel of these lines to study potential impacts of APP dosage or structure on two distinct steps of neurogenesis in trisomic cells: 1) terminal differentiation of human neuro-progenitor cells (NPC) to post-mitotic neurons and 2) neuron structure as reflected in neurite outgrowth. Complete loss of APP causes marked defects in each of these two distinct steps, reducing both the terminal differentiation of NPCs to neurons, and proper neurite development for extended neuron structure. Hence, APP is necessary for both aspects of normal neurogenesis. Further analyses of the null and other mutant lines indicate that APP likely impacts these two distinct steps by two different mechanisms. Collective results suggest that the reduced terminal differentiation of NPCs reflects an effect of APP dosage, whereas the defects in neurite extension are due to structural mutation of the APP extracellular domain. Absence of APP or reduced (monosomic) APP dosage prolonged the cycling of trisomic NPCs, which is known to be regulated by Notch signaling. APP and Notch are the main targets of gamma-secretase cleavage, hence we hypothesized that APP dosage may impact neurogenesis indirectly, potentially via effects on Notch signaling. To test this, we treated NPCs with Compound E which inhibits gamma-secretase (and Notch signaling); results show this restored levels of neurogenesis in APP depleted lines, supporting an indirect effect of APP dosage . In contrast, results indicate that disruption of APP extracellular domain integrity impacts neurite extension via a more direct role of APP in neuron structural maturation. This study describes a resource of well-characterized APP mutant isogenic DS iPSC lines, implicates a dynamic interplay between APP dosage and Notch signaling, and raises new questions about the impact of APP dosage in orchestrating neural progenitor fate decisions during human brain development, specifically in the context of trisomy 21.

ID: 42624823
Title: Partial deletion in the cuticular protein gene BmorCPR2 results in a body shape mutant in silkworm, Bombyx mori L. (Lepidoptera: Bombycidae).
Abstract: The Bombyx mori L. (Lepidoptera: Bombycidae) is a significant economic insect used for silk production. A novel body shape mutant, stony^sunken (st^sk), that exhibits a sunken intersegmental membrane was isolated from the wild type of st^sk (WT-n08). Investigation indicated that the mutation had no significant effect on its growth and development. To elucidate the molecular mechanism underlying this body shape mutant, genetic analysis, positional cloning, and the CRISPR/Cas9 gene editing system were performed. Genetic analysis demonstrated that the mutant trait in st^sk is controlled by an autosomal recessive gene and follows Mendelian inheritance. Positional cloning showed that a putative cuticular protein gene, BmorCPR2 on chromosome 8, was the candidate gene. Sequencing analysis revealed partial deletion of BmorCPR2 exon 2 and intron 2 sequences occurred and subsequently resulted in the premature termination of gene expression. Knock-out of BmorCPR2 using the CRISPR/Cas9 gene editing system led to the sunken intersegmental membrane phenotype. These findings highlight the essential role of BmorCPR2 in silkworm cuticular formation, providing a foundation for further research on cuticular protein function.

ID: 42625114
Title: Molecular pathways and emerging therapeutic strategies in advanced thyroid cancer: from targeted therapy to precision oncology.
Abstract: The most prevalent endocrine cancer is thyroid cancer. It may act in a very disparate manner; ranging between slow growing, well differentiated tumours to an aggressive anaplastic subtype. Initial treatment with standard therapy, surgery, radioactive iodine, the thyroid-stimulating hormone inhibition and in some cases, chemotherapy or external-beam radiations are effective with the differentiated cancers at the early stages. Nevertheless, there is no choice with tumours, which become unresponsive to iodine, metastasized, or un- differentiated. Recent developments in molecular oncology have elucidated the genetic alterations that contribute to thyroid cancer, in particular, the MAPK and PI3K/AKT/mTOR pathways, and BRAF, RAS, RET, and TERT promoter mutations. The current review summarizes the changing therapeutic environment of thyroid cancer that includes established targeted medicines and novel developments. These are immune checkpoint inhibitors, redifferentiation based on reinstatement of iodine uptake, selective RET and BRAF/MEK, RNA-based therapeutics, CRISPR gene editing, oncolytic virotherapy, peptide receptor radionuclide therapy, alpha-particle radiotherapy, and nanotechnology-based drug delivery systems. A combination regimen involving molecular targeting and immunotherapy or radioactive iodine is also improving the disease in advanced disease. Collectively, the advances are an indication of a transition to the personalized, mechanism-driven treatment strategies that would prolong the survivability of patients with advanced thyroid cancer, conquer resistance, and reduce systemic toxicity.

ID: 42628204
Title: Blocking the conversion of β-carotenoids into xanthophylls through HYb gene editing via CRISPR/Cas9 confers enhanced heat stress tolerance in Nicotiana tabacum.
Abstract: Plants produce protective metabolites to withstand stress, and β-carotenoids act as crucial antioxidants. β-Carotene is converted into xanthophylls by β-carotene hydroxylase (BCH). In this study, the BCH gene in Nicotiana tabacum was targeted using CRISPR/Cas9 genome editing to redirect metabolic flux toward β-carotene accumulation and evaluate its effects on heat tolerance. Following Agrobacterium-mediated transformation with a BCH-specific gRNA, three mutant plants were obtained; two carrying frameshift mutations (P1 and P3) and one with mis-sense mutations (P2). A significant increase in chlorophyll contents was observed in the edited lines compared with wild-type plants. Interestingly, the frameshift mutants exhibited substantial increase in total chlorophyll (71% and 31% in P3 and P1 respectively) as compared to mis-sense mutant P2 (18%). Further, biochemical profiling using HPLC showed that the edited plants accumulated 3.74-fold increase in β-carotene than wild-type plants. Under heat stress (42 °C for 96 h), the edited plants exhibited enhanced thermotolerance by remaining greener and delayed wilting, whereas wild-type plants developed necrosis rapidly under heat stress. This enhanced thermotolerance might has resulted from the increased expression of ε-cyclase (an upstream key enzyme in β-carotene biosynthesis) as revealed by expression profiling of edited plants. In contrast, the expression of downstream β-cryptoxanthin and zeaxanthin biosynthetic genes was reduced, which seems consistent with mutation of BCH. DAB staining further confirmed reduced ROS accumulation in edited plants as compare to wild type supporting the protective role of β-carotene. In summary, the downregulation of BCH in N. tabacum boosted β-carotene levels and chlorophyll retention, enhancing heat stress tolerance. These findings demonstrate the potential of targeting carotenoid biosynthesis to improve crop resilience.

ID: 42630992
Title: Regulating human reproductive and developmental biotechnologies: a UK reference model.
Abstract: Rapid scientific change is exposing the limits of the UK's regulation of human reproductive and developmental biology. Embryo-like structures, in vitro gametogenesis, organoids and heritable genome editing advance understanding of human development and physiology and support new applications in assisted reproduction and clinical care, yet the current regulatory framework is ill-equipped to govern them coherently. A case is made for coordinated, technology-neutral regulatory change. The article proposes the principles of a model UK Human Reproductive and Developmental Biotechnology Act and regulator, the Human Reproductive and Developmental Biotechnology Authority-or HRBA, pronounced 'harbor'-to provide a permissions route across research and translation. The model Act is a modular, risk-tiered scheme with consistent standards and tailored red lines. It is designed to accommodate boundary cases, to integrate and build upon the existing remit of the Human Fertilisation and Embryology Authority, and coordinate with other established regulators to retain institutional expertise and avoid unnecessary duplication. The proposal is ambitious and would help give effect to UK Government commitments to safely advance the life sciences in this area. But even if piecemeal amendments are ultimately favored, it clarifies the underlying choices and trade-offs and provides a benchmark against which changes can be tested.