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PubMed2026

Stable simulations do not guarantee functional engagement: a case study of off-target prediction for Seladelpar and Zanamivir.

Identifying off-target interactions of approved drugs is important to anticipate side effects and uncover repurposing opportunities. Computational pipelines combining structural homology, structure prediction, and molecular dynamics (MD) simulations offer a promising strategy, but it remains unclear whether stable, control-like MD trajectories reliably indicate functional engagement. We examined this in a case study of two approved drugs. Using the Evolutionary Classification of Protein Domains (ECOD) framework to select candidate off-targets, we modeled each drug-protein complex as two independent AlphaFold3 models and simulated both by MD, for Seladelpar (a PPARδ agonist) and Zanamivir, an influenza neuraminidase inhibitor that also inhibits human Sialidase-2 (NEU2). Candidates were ranked by the similarity of global MD descriptors to the on-target control. For Seladelpar, the three top-ranked candidates (FXR, RARγ, ERRγ) were tested experimentally; the Zanamivir set was analyzed computationally only. None showed measurable activity in reporter or thermal shift assays, despite stable simulations and descriptor values comparable to the control. Including PPARα and PPARγ as weak-positive comparators, these descriptors did not rank genuine interactions closer to the control than inactive candidates. Residue-level comparison with experimental structures showed the predicted poses reproduced only part of the canonical contacts. Where experimental drug-bound structures existed, AlphaFold3 reproduced the pose for PPARα but not PPARγ, and its per-model confidence did not track pose accuracy. Within this case study, the specific global descriptors examined reflect complex stability rather than functional engagement, which does not mean MD-based approaches cannot make this distinction.

Zanamivir↗

PubMed2026

Suppression of HIV-1 replication in CEM-A cell cultures by trans-splicing group I introns targeting PAS/PBS sequences and conditionally expressing ΔN-Bax.

Anti-HIV group I introns containing antisense guide sequences directed against the HIV-1 primer activation signal and primer-binding site (PAS/PBS) were designed and evaluated. Because PAS/PBS sequences are present in the viral RNA species examined, these RNAs can serve as trans-splicing substrates. The introns were active against both artificial target RNAs and viral RNA generated during infection. Cleavage and degradation of targeted viral RNA may have contributed to suppression, whereas inclusion of a 3' exon encoding the proapoptotic protein ΔN-Bax was associated with increased programmed cell death and may have augmented suppression of viral replication. In cultured CEM-A cells, transgene expression of these introns markedly suppressed HIV-1 replication, with p24 levels falling below the assay detection limit in selected clones. RESULTS: RT-PCR and sequence analysis detected splice products containing the expected PAS/PBS junctions. In the dual-luciferase assay, intron expression reduced normalized Gaussia luciferase signal by approximately 70% relative to the negative control. Qualitative Annexin V imaging and caspase-3 assays were consistent with infection-dependent apoptosis after ΔN-Bax splice-product formation. Transient expression of each intron in HEK293T cells followed by infection with VSV-G-pseudotyped HIV-1NL4-3 at an MOI of 2 reduced p24 levels by approximately 50% at 4 days post-infection. Construct 128L produced the strongest RT-PCR band under the tested conditions and was selected for subsequent experiments. A canonical splice product and a low-abundance noncanonical splice product were detected; both involved the intended HIV-derived target RNA, although transcriptome-wide off-target splicing was not assessed. Heterogeneous transformed HEK293T populations showed an approximately 2-log10 reduction in p24. In selected clonal HEK293T and CEM-A lines, p24 was below the assay detection limit at the measured endpoints, including up to 90 days after infection in some CEM-A clones. CONCLUSIONS: PAS/PBS-targeting group I introns suppressed HIV-1-associated p24 production in the tested cell-culture models. Linking the introns to a ΔN-Bax 3' exon was associated with infection-dependent apoptosis and may further limit viral replication and spread. The use of highly conserved, functionally constrained target sequences may reduce the likelihood of escape, but viral evolution and transcriptome-wide off-target effects were not assessed. This conditional death-upon-infection strategy warrants further evaluation in primary-cell and in vivo models.

Humans↗

PubMed2026

Emerging techniques of CRISPR/Cas system in antiviral therapy and diagnostics: Applications, limitations, and translational perspectives.

The CRISPR/Cas (clustered regularly interspaced short palindromic repeats) system is a versatile technology for developing antiviral medicines and editing viral genomes in both diagnostics and vaccine synthesis. Emerging insights into class 2 effectors, such as Cas9, Cas12, and Cas13, which target viral DNA and RNA, have revolutionized vaccines against viruses such as HIV, HPV, HBV, and EBV. Innovative diagnostic techniques such as SHERLOCK, DETECTR, and FELUDA have demonstrated system's diversity and accuracy in detecting the virus markers, supporting clinical decision-making, indicating adaptability and precision of CRISPR. This review critically evaluates CRISPR's role in RNA editing, emphasizing its importance for functional genomics and development of recombinant vaccines. Translational challenges are critically discussed, including off-target effects, delivery limitations, and ethical issues, for which unique approaches such as high-fidelity Cas variants, non-viral delivery systems, and bioethical frameworks are evaluated to address these limitations. This review also covers other social implications, such as accessibility and biosecurity risks, associated with CRISPR technologies Collectively, these advances underscore the transformative potential of CRISPR technologies in shaping next-generation antiviral diagnostics and therapeutics.

CRISPR-Cas Systems↗

PubMed2026

Synergistic HMGN1 and VP64 Fusions Potentiate High-Precision and PAM-Flexible Base Editing.

RNA-guided CRISPR-derived base editors (BEs) have revolutionized genome editing by enabling targeted base substitutions. However, their application is frequently constrained by the stringent requirement for PAM sequences and low editing precision (bystander editing). Here, we present a robust strategy to overcome these limitations by coupling SpRY, a near-PAM-less Cas9 variant, with truncated CDA1 cytidine deaminases. While this combination enables precise editing of virtually any cytosine in the genome, it initially exhibited suboptimal efficiency. To address this, we systematically screened a diverse panel of candidate DNA-binding proteins and identified that the synergistic fusion of HMGN1 and VP64 substantially enhances editing activity without compromising precision. Importantly, this enhanced editing efficiency was achieved without markedly increasing off-target effects. Our new BEs demonstrated robust performance not only in yeast but also in rice, suggesting broad applicability in gene therapy, precision breeding, and fundamental research.

Gene Editing↗

PubMed2026

CRISPR-Engineered CAR-T Cell Therapy for Epstein-Barr Virus-Associated Nasopharyngeal Carcinoma: A Review of Emerging Therapeutic Prospects.

Epstein-Barr virus (EBV)-associated nasopharyngeal carcinoma (NPC) remains a clinically challenging malignancy, particularly in recurrent or metastatic disease where durable responses to chemoradiotherapy and immune checkpoint blockade are limited. The viral aetiology of NPC provides a strong biological rationale for immune-based treatment; however, translation of chimaeric antigen receptor (CAR) T-cell therapy into this solid tumour setting is constrained by poor tumour trafficking, antigen heterogeneity, limited surface accessibility of EBV latent antigens, T-cell exhaustion, and an immunosuppressive tumour microenvironment. This review critically evaluates the emerging therapeutic prospects of CRISPR-engineered CAR-T cell therapy for EBV-associated NPC. It synthesises evidence on EBV latency biology, NPC immune evasion, solid-tumour CAR-T limitations, and genome-engineering strategies including conventional CRISPR-Cas9, base editing, prime editing, and double-strand-break-sparing targeted integration. Particular attention is given to genotoxicity, chromosomal rearrangements, chromosome loss, bystander and off-target editing, manufacturing heterogeneity, and the regulatory and biological barriers that currently separate technical feasibility from NPC-specific clinical implementation. Available clinical evidence from checkpoint blockade, EBV-specific adoptive T-cell therapy, base-edited CAR-T cells in haematologic malignancy, and early CRISPR-edited T-cell trials supports the feasibility of immune and genetic redirection but does not establish efficacy of a clinically validated CRISPR-engineered CAR-T platform for NPC. Future development should prioritise surface-accessible antigen validation, fit-for-purpose selection of editing technology, genomic safety, scalable manufacturing, and biomarker-driven early-phase trials.

Humans↗

PubMed2026

CRISPR-Cas and Infectious Diseases: A Decade of Translational Advances in Molecular Biotechnology.

CRISPR-Cas systems have emerged as a versatile tool for diagnosing, treating, and preventing infectious diseases. This review highlights translational advancements in CRISPR-Cas-based applications, concentrating on the past decades in diagnostics, therapeutic genome editing, and vaccine development. The article highlights key platforms like DETECTR and SHERLOCK, which enable rapid, sensitive pathogen detection, and explores CRISPR-Cas9 systems in therapeutic strategies for directly targeting viral genomes and combating antimicrobial resistance. It also examines the role of CRISPR-Cas9 in engineering live-attenuated and personalized neoantigen vaccines. Principal findings demonstrate a clear progression from experimental proof-of-concept to preclinical applications primarily in CRISPR-based diagnostics and the engineering of live-attenuated vaccine candidates, whereas translation in CRISPR-based therapeutics and personalized neoantigen vaccines for infectious diseases remains at earlier, more exploratory stages. CRISPR-based diagnostics have progressed further toward clinical evaluation than therapeutics due to delivery and safety constraints, while personalized neoantigen vaccines are included mainly as an emerging, comparative concept for infectious diseases rather than a mature application. This review uniquely integrates CRISPR-based diagnostics, therapeutics, and vaccine development within a single infectious disease framework, critically assesses their current maturity, and systematically highlights technical, regulatory, and ethical barriers alongside realistic future priorities. The review concludes that while CRISPR-Cas holds transformative potential for infectious disease management, significant challenges in delivery efficiency, off-target effects, and ethical regulation must be addressed to ensure safe and equitable clinical translation.

Humans↗

PubMed2026

From Gene Function to Precision Intervention: CRISPR/Cas9 and Stem Cell-Based Strategies as Emerging Disease-Modifying Approaches in PMOS.

Polyendocrine metabolic ovarian syndrome (PMOS) is a complex endocrine-metabolic disorder affecting up to 18% of women worldwide and remains the leading cause of anovulatory infertility. Despite extensive research, current treatments primarily target symptoms, including menstrual irregularities, hyperandrogenism, and metabolic dysfunction, without addressing the underlying molecular and tissue-level disturbances. Advances in multi‑omic profiling have identified disruptions across neuroendocrine, metabolic, inflammatory, and extracellular matrix pathways, alongside genetic susceptibility at loci such as DENND1A, CYP17A1, LHCGR, FSHR, IRS1, and PPARG. However, the functional roles of many variants remain unresolved. CRISPR/Cas9 gene editing enables precise interrogation of these pathways, while stem cell-based platforms, including mesenchymal stem cells (MSCs), exosomes, and gene-edited induced pluripotent stem cells (iPSCs), may serve as complementary platforms for regeneration and disease modeling. Preclinical studies demonstrate that MSCs and their derivatives modulate inflammation, restore ovarian structure, and improve metabolic parameters, while iPSC-based models enable patient-specific investigation of steroidogenic and metabolic abnormalities. Translational challenges remain, including targeted delivery, off-target effects, phenotypic heterogeneity, and regulatory considerations. Integrating CRISPR‑based functional genomics with stem cell research may shift PMOS management from symptom‑focused care to targeted, mechanism‑driven interventions that could modify the course of PMOS (Graphical Abstract).

Humans↗

PubMed2026

Discovery of NAT-6-321056 as a novel modulator of VEGFR2 signaling to suppress tumor angiogenesis.

Vascular endothelial growth factor receptor 2 (VEGFR2) is a master regulator of angiogenesis and cancer progression. However, current VEGFR2 modulators face significant challenges, including off-target toxicity and acquired resistance, underscoring the urgent need for novel therapeutic agents with improved efficacy and safety profiles. Here, we reported that virtual screening of 39,442 natural products from the ZINC natural products-derived library, coupled with molecular docking and molecular dynamics (MD) simulations to evaluate the binding stability of candidate compounds, identified NAT-6-321056 as a highly promising modulator of VEGFR2 signaling. Biological evaluations demonstrated that NAT-6-321056 exerted potent inhibition on the growth of a broad spectrum of cancer cells, including both solid tumors and hematological malignancies. In EA.hy 926 endothelial cells and SK-N-DZ neuroblast cells, the compound significantly suppressed proliferation, migration, and invasion. Microscale thermophoresis (MST) confirmed direct binding of NAT-6-321056 to VEGFR2 with favorable affinity. Kinase profiling against a panel of 33 kinases indicated that NAT-6-321056 exhibited a multi-kinase modulation profile. Mechanistic studies revealed that NAT-6-321056 suppressed the expression of hypoxia-inducible factor 1-alpha (HIF-1α) and was associated with reduced VEGFR2 phosphorylation and attenuation of the downstream ERK/JNK/AKT signaling pathways. Moreover, NAT-6-321056 exhibited robust in vivo anti-angiogenic effects in both the chick chorioallantoic membrane (CAM) assay and transgenic zebrafish vascular fluorescence imaging models. Computational absorption, distribution, metabolism, excretion, and toxicity (ADMET) prediction suggested acceptable drug-like properties. Collectively, these findings demonstrated that NAT-6-321056 is a promising modulator of VEGFR2 signaling with potent anti-angiogenic activity and represents a viable candidate for cancer therapy.

Vascular Endothelial Growth Factor Receptor-2↗

PubMed2026

Cre-loaded integrase-defective lentiviral vectors for targeted cassette exchange in CHO cells.

Genome-modifying enzymes, such as recombinases and CRISPR-associated nucleases, enable targeted gene insertion when delivered transiently to minimize off-target effects. Precise genome engineering requires controlled enzyme activity, as well as efficient donor DNA transfer. Integrase-defective lentiviral vectors (IDLVs) provide a promising platform for transient episomal DNA transfer; however, their integration efficiency depends on complementary genome-targeting strategies. Here, we engineered Cre-loaded IDLVs (Cre-IDLVs) that co-package lentiviral vector genomes together with bioactive Cre recombinase. Cre was inserted into the Gag region of an integrase-defective gag-pol construct, allowing for efficient encapsidation and protease-mediated release during virion maturation without compromising the viral titer. The resulting particles carried donor cassettes flanked by heterospecific loxP sites. When applied to CHO founder cells harboring compatible genomic loxP landing pads, Cre-IDLVs efficiently mediated recombination-mediated cassette exchange, producing the highest number of G418-resistant colonies among the plasmid ratios tested. Genomic PCR and sequencing confirmed precise locus-specific insertion without detectable random integration in the analyzed clones. These findings establish Cre-IDLVs as a streamlined dual-delivery platform that couples transient recombinase activity with episomal donor DNA transfer. This hybrid lentiviral strategy provides a programmable approach for controlled and site-specific genome modification in mammalian cells.

Integrases↗

PubMed2026

Isolation of region-specific factors driving antibody class-switch recombination from the immunoglobulin heavy chain locus.

Activation-Induced Cytidine Deaminase (AID) induces DNA double-strand breaks (DSBs) at the switch (S) regions of the Immunoglobulin heavy chain (IgH) locus, which are essential for class switch recombination (CSR) and somatic hypermutation (SHM), key processes for effective antibody production. While AID activity is critical, its off-target effects, such as DSBs at the Myc locus, can cause chromosomal translocations like IgH-Myc fusions, contributing to B-cell lymphomas. The factors assembled on the IgH locus that help restrict AID-induced DSBs and subsequently CSR, remain unknown. To address this, we developed a method to isolate CSR-specific factors by inserting a 5×-GAL4-UAS sequence at the switch-mu (Sμ) region in CH12 cells. This engineered site enables recruitment of a 3-FLAG-GAL4 DNA-binding protein (3F-GAL4-DBD), allowing specific pulldown of proteins enriched at the Sμ region. Successful recovery of the known CSR regulator BRD2 from the Sμ region, along with enrichment of the DNA repair factors 53BP1 and gH2AX, validated this approach. Identification and characterization of IgH-enriched factors establish a validated methodological framework to facilitate future proteomic discovery of CSR regulators and highlight mechanisms that balance antibody diversification with genomic integrity in B cells.

Immunoglobulin Class Switching↗

PubMed2026

Engineering extracellular vesicles for targeted siRNA delivery: Advances, therapeutic applications, and clinical translation.

Small interfering RNA (siRNA) therapeutics have emerged as a transformative approach for sequence-specific gene silencing, offering the potential to treat a broad spectrum of diseases by selectively suppressing disease-associated genes. However, the clinical translation of siRNA remains limited by rapid enzymatic degradation, poor cellular uptake, inadequate endosomal escape, and off-target effects, necessitating the development of efficient delivery systems. Extracellular vesicles (EVs) have gained considerable attention as natural nanocarriers owing to their excellent biocompatibility, low immunogenicity, intrinsic targeting capability, and ability to protect therapeutic cargo while traversing complex biological barriers. This review comprehensively discusses the biological characteristics of EVs, the molecular basis of RNA interference, and the major challenges associated with siRNA delivery [Fig. 1]. Recent advances in EV engineering, including cargo-loading strategies such as electroporation, sonication, extrusion, parent-cell engineering, and microfluidic approaches, together with surface functionalization using peptides, antibodies, aptamers, and hybrid nanoplatforms, are critically evaluated for improving targeting specificity and intracellular delivery. Furthermore, the therapeutic applications of engineered EV-mediated siRNA delivery in cancer, neurological disorders, liver diseases, cardiovascular diseases, inflammatory disorders, and infectious diseases are systematically summarized, highlighting their potential to enhance gene silencing while minimizing systemic toxicity. Current challenges related to large-scale manufacturing, cargo-loading efficiency, standardization, quality control, regulatory approval, and clinical translation are also discussed, together with emerging technologies involving synthetic biology, genome engineering, artificial intelligence, and multifunctional hybrid vesicles. Overall, engineered extracellular vesicles represent a highly versatile and biologically inspired platform for targeted siRNA delivery, providing a promising foundation for the development of next-generation precision RNA therapeutics and accelerating the clinical translation of gene-silencing strategies.

Extracellular vesicle engineering↗

PubMed2026

Shared ligand-blocking mechanism but distinct conformational modulation by α5-targeting antibodies BIIG2 and MINT1526A.

Integrins are heterodimeric receptors important for cell adhesion and signaling. Integrin α5β1 is a key mediator of angiogenesis and its dysregulation is associated with tumor progression and metastasis. Despite numerous efforts, α5β1-targeting therapeutics have been unsuccessful due to poor efficacy and off-target effects. A contributing factor is our limited understanding of how integrin conformation influences interactions with therapeutics. Using cell-based functional assays, patient-derived xenografts, biophysics, X-ray crystallography, and electron microscopy, we shed light on these relationships by characterizing two anti-α5β1 antibodies, BIIG2 and MINT1526A. We show that both antibodies bind α5β1 with nanomolar affinity, reduce tube formation in vitro, and bind overlapping epitopes that block fibronectin binding. However, using electron microscopy, we reveal that while BIIG2 binding does not substantially alter the conformational states, MINT1526A preferentially recognizes the bent conformation and restricts the conformational ensemble. These insights can guide which aspects to prioritize to improve the design of future integrin-targeted therapeutics.

angiogenesis↗

PubMed2026

Efficient and precise programmable DNA knock-in without double-strand breaks.

Programmable gene knock-in holds substantial promise for treating genetic diseases and advancing cell therapies. However, achieving precise and efficient kilobase-scale DNA fragment integration remains challenging1,2. Here we report CRISPR kilobase-scale nickase-targeting (KNIT) editing for efficient, precise and programmable kilobase-scale DNA insertion without double-strand DNA cleavage, which is enabled through the coupling of a Cas9 nickase with a DNA donor recruiting system. KNIT editing facilitates programmable integration of DNA fragments from 0.7 kb to more than 10 kb and is effective across genomic loci and cell types. It achieves up to 89% efficiency and markedly reduces unintended insertion-deletion mutation (indels) rates, translocations and off-target editing. The system supports repeated insertion editing and multiloci gene knock-in with minimal translocations. Its enhanced version, KNIT editor 2, further improves efficiency via a single transfection. Moreover, in mutant cells with a pathological mutation, KNIT editing restores normal gene expression by inserting a therapeutic gene into a safe harbour locus or its native locus. Notably, KNIT editing enables non-viral and programmable chimeric antigen receptor T cell (CAR-T cell) engineering without double-strand breaks and with clinically relevant efficiencies. Moreover, the engineered CAR-T cells exhibit effective antitumour activity in vitro and in mouse models. Therefore, by achieving programmable and site-specific kilobase-scale DNA insertions without double-strand breaks while reducing unintended outcomes, KNIT editing provides a versatile platform for advancing personalized medicine.

Animals↗

PubMed2026

Single-cell profiling of mitochondrial phenotyping-coupled mtDNA genotyping.

Simultaneously profiling mitochondrial DNA (mtDNA) heteroplasmy and phenotypic variability at the single-cell level remains a challenge due to the absence of integrated methods that map mitochondrial genotypes alongside their functional states. We introduce human single-cell mitochondrial phenotype-coupled mtDNA sequencing (scMPCDS), a platform that quantifies mtDNA mutations and heteroplasmy together with mitochondrial membrane potential and reactive oxygen species within individual cells. Unlike bulk sequencing or separate single-omics techniques, scMPCDS directly correlates mitochondrial genomic instability with functional outcomes. Using this approach, we demonstrate that DdCBE-mediated mtDNA editing induces cell-specific off-target mutations in the mitochondrial genome, which coincide with diverse phenotypic changes. Applying scMPCDS to HeLa cells and clear cell renal cell carcinoma tissues, we identify single-cell subpopulations exhibiting distinct mtDNA mutation burdens and altered bioenergetic profiles, implicating potential mitochondrial heterogeneity-driven tumor evolution. Overall, scMPCDS serves as a versatile tool to unravel mitochondrial genotype-phenotype relationships at the single-cell level in both normal and disease states, thereby advancing precise mitochondrial diagnostics and therapeutics.

Humans↗

PubMed2026

SURE-Pipe: a pipeline to compare genomes and extract shared and unique regions.

Identification of unique and shared genomic regions between organisms has substantial translational potential for the development of marker-based diagnostic assays and sequence homology-driven taxonomic classification. An automated pipeline capable of performing genome comparisons at both the intra- and inter-species levels with minimal computational requirements can significantly advance genome-driven translational research. Species-specific genomic regions are particularly valuable for sequence-based species identification and for developing DNA amplification- or hybridization-based diagnostic assays. Here, we present SURE-Pipe, an automated and flexible pipeline for genome comparison and extraction of unique and shared genomic regions (https://github.com/BPaul-bioinfoLAB/SURE-Pipe). Benchmarking of this pipeline using simulated datasets demonstrated high accuracy for shared and unique region identification. Using the pairwise genome comparison module, six genome pairs from diverse microorganisms were analysed, and identified the unique and shared regions. In addition, the multigenome comparison module was applied to 96 genomes representing 24 Bacillus species and identified species-specific genomic regions. These regions were highly conserved among four strains of a species (>98% sequence identity) and exhibit little to no similarity with other species. Species-specific primers designed for all 24 Bacillus species showed no off-target amplification in in-silico polymerase chain reaction analysis, indicating their specificity. Overall, SURE-Pipe provides a robust and multipurpose framework for comparative genomics, and the outcomes can be used for species identification and the development of genome-based diagnostic approaches.

Genome, Bacterial↗

PubMed2026

Development of a Multiplex Polymerase Chain Reaction Assay for Differentiating Three Lactococcus Species Associated With Piscine Lactococcosis.

Piscine lactococcosis is an important bacterial disease of farmed fish. The causative agents, Lactococcus garvieae, Lactococcus petauri and Lactococcus formosensis, are closely related, which complicates species-level identification. We developed a conventional multiplex PCR assay targeting species-specific genes identified by comparative genomic analysis. Average nucleotide identity reassignment of 441 publicly available genome assemblies identified 111 L. garvieae, 255 L. petauri and 75 L. formosensis genomes. Species-specific primers and a tuf-based Lactococcus common control were evaluated using in silico polymerase chain reaction (PCR) against target genomes and 10,460 off-target assemblies representing 474 taxa in 12 genera. Experimental specificity was assessed using six target strains and 19 non-target fish pathogens. Distinct amplicons of 195, 333 and 500 bp were produced for L. garvieae, L. petauri and L. formosensis, respectively, together with a 132-bp control amplicon. No cross-amplification was observed. All target species were detected in mixed-DNA samples and spiked kidney and spleen homogenates from two fish species. Analytical detection limits were estimated based on microscopic cell counts of bacterial suspensions before DNA extraction and ranged from 0.956 to 8.55 cell equivalents per reaction. This assay represents a rapid, low-cost method for differentiating lactococcosis-causing Lactococcus species using standard PCR and agarose gel electrophoresis.

Lactococcus garvieae↗

PubMed2025

CRISPR RNP-Mediated Transgene-Free Genome Editing in Plants: Advances, Challenges and Future Directions for Tree Species.

CRISPR ribonucleoprotein (RNP)-mediated genome editing offers a transgene-free platform for precise genetic modification in diverse herbaceous and tree species, including rice, wheat, apple, poplar, oil palm, rubber tree and grapevine. However, its application in woody plants faces distinct challenges, notably inefficient delivery and regeneration difficulties, particularly in species such as bamboo. While some of these issues also occur in herbaceous plants, they are often significantly more complex in woody species due to factors such as intricate cell wall architecture, widespread recalcitrant genotypes and inherent limitations of current delivery platforms. This review presents the first in-depth, critical re-evaluation of recent advancements in RNP-mediated editing in woody plants, highlighting these obstacles that warrant focused attention. Unlike plasmid-based CRISPR systems, RNP editing utilises Cas9/Cas12a protein-guide RNA complexes without integrating foreign DNA. This enables a DNA-free editing strategy that simplifies regulatory approval and minimises off-target effects due to the transient presence and rapid degradation of RNPs within plant cells. While PEG-mediated protoplast transfection and particle bombardment remain the primary reported methods for RNP delivery in trees, we evaluate promising alternative strategies such as lipofection, electroporation, cell-penetrating peptides and nanoparticle-based systems for targeted RNP delivery. Despite their promise, these advanced methods remain largely untested in woody species. Finally, we outline future research directions, including the development of tree-specific RNP delivery systems and regeneration protocols to enhance efficiency and minimise cytotoxicity. These innovations are essential for unlocking the full potential of RNP-mediated genome editing in long-lived tree species. This review provides a focused and timely roadmap for expanding the application of RNP technology across diverse woody plants.

Gene Editing↗

PubMed2026

Gene therapy for genodermatoses at the crossroads of innovation and clinical translation.

Inherited genodermatoses are a heterogeneous group of rare monogenic disorders. Among these, epidermolysis bullosa (EB) and ichthyoses represent paradigmatic disorders characterized by severe skin fragility and hyperkeratosis, respectively, and impaired barrier function, often with profound effects on quality of life and systemic health. Current management remains largely palliative, underscoring the urgent need for disease-modifying therapies. Over the past 2 decades, advances in epithelial stem cell biology, vector engineering and genome editing technologies have transformed the therapeutic landscape for genodermatoses. Ex vivo gene therapy has provided the first proof that genetically corrected epidermal stem cells can achieve long-term tissue regeneration in EB skin patients, establishing a new paradigm for regenerative medicine. In parallel, the emergence of programmable genome engineering platforms, including CRISPR/Cas nucleases, base editors and prime editors, have enabled increasingly precise strategies for mutation-specific correction in both recessive and dominant disorders. Furthermore, the development of in vivo topical approaches is expanding the possibility of directly targeting the skin. Despite these advances, substantial translational barriers continue to limit broad clinical implementation. Efficient and durable targeting of epidermal stem cells within a highly regenerative tissue, together with safe delivery across the skin barrier, stringent control of off-target activity, scalable manufacturing and demonstration of long-term safety, remain major challenges for the clinical translation of these approaches. In this Review, we discuss the current state of gene therapy for genodermatoses, highlighting key clinical milestones, emerging genome editing technologies and next-generation delivery systems. We further examine the biological and regulatory challenges that need to be overcome to bridge the gap between experimental innovation and clinically accessible therapies for patients with inherited skin diseases.

epidermolysis bullosa (EB)↗