Search Results (2,768)

Allogeneic T cell therapies have emerged as a promising strategy to overcome the logistical and manufacturing limitations of autologous approaches, enabling scalable, off-the-shelf cancer immunotherapy. While early clinical efforts have focused predominantly on αβ T cell-based platforms, including CAR- and TCR-engineered approaches, a growing spectrum of alternative cell types, such as γδ T cells, invariant natural killer T cells, mucosal-associated invariant T cells, and induced pluripotent stem cell-derived effectors, is expanding the design landscape of allogeneic therapies. However, clinical translation remains constrained by immune rejection, limited persistence, lymphodepletion-associated toxicity, manufacturing variability, and impaired efficacy in solid tumors. To address these barriers, engineering strategies have increasingly integrated T cell receptor disruption, human leukocyte antigen modulation, cytokine support, checkpoint editing, and synthetic circuit design. This review provides an oncology-focused, cross-platform framework for evaluating diverse allogeneic T cell and T cell-like platforms according to clinical maturity, safety, manufacturability, persistence, and tumor-targeting capacity. We further discuss how platform-specific biological properties and clinical evidence can be integrated with modular engineering strategies to optimize antitumor performance. These insights support a shift from platform-centric development toward a design-driven paradigm for next-generation allogeneic cellular immunotherapies with improved efficacy, safety, and scalability. Full article

Background: Lychee fruits are sweet and juicy, yet mitochondrial genomic data for this species remains scarce, limiting in-depth studies of its genetic and evolutionary characteristics. To address this gap, in this study, the abortive-seeded cultivar ‘Xianjinfeng’ (XJF) and the large-seeded cultivar ‘Xinqiumili’ (XQML) were selected for analysis. Using third-generation sequencing technology, we sequenced, assembled, and annotated their mitochondrial genomes, and compared their structural characteristics and evolutionary relationships. Results: Assembly revealed mitochondrial genome sizes of 579,270 bp for XJF and 579,261 bp for XQML, both with 45.41% GC content. The mitogenomes contain 396 repetitive sequences, including 47 tandem repeats and 165 dispersed repeats, with SSR loci primarily 10–14 bp in length. Each genome encoded 62 genes, comprising 22 tRNAs, 3 rRNAs, and 35 protein-coding genes. Further analysis revealed 15 homologous sequences originating from chloroplasts in both mitochondrial genomes, totaling 12,194 bp (2.11% of the mitochondrial genome). These included 9 tRNA genes, 4 rRNA genes, and partial protein-coding sequences. Additionally, 184 simple sequence repeats (SSRs) were identified in both cultivars, whereas 564 and 563 potential RNA editing sites were predicted by computational tools in XJF and XQML, respectively, indicating subtle genetic differences between the cultivars. This study also analyzed codon usage preferences, nucleotide diversity, and chloroplast-to-mitochondria gene transfer events. Collinearity and comparative genomics results indicate that lychee is closely related to Nephelium lappaceum L. and Xanthoceras sorbifolium Bunge within the Sapindaceae family. Conclusions: In this study, two high-quality lychee mitochondrial genomes were successfully assembled and annotated, enriching the mitochondrial genome resources of Sapindaceae plants and laying a foundation for future lychee phylogenetic and evolutionary studies of closely related species. Full article

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. Full article

CRISPR-Cas9 has transformed microbial biotechnology by enabling precise genome modifications; however, achieving high editing efficiency remains a challenge due to multiple determinants, including on-target specificity, off-target events, PAM sequence, sgRNA scaffold composition, and RNA secondary structure. Our review foresees how artificial intelligence (AI) can address those challenges by enabling automated identification as well as highly active guide RNA (gRNA) optimisation. We highlight the influence of a data-driven training strategy that is focused on high-quality, diverse, and accurately labelled microbial datasets—mainly, given the limitations of models derived from mammalian systems that are not directly transferable to microbial organisms. Moreover, we discuss the key role of FAIR (Findable, Accessible, Interoperable, and Reusable) data principles and centralised, curated CRISPR-Cas databases as foundational elements for developing robust and predictive frameworks. Emerging directions are also explored, including generative AI approaches capable of supporting automated experimental planning. By considering the potential dual use of such technologies, the review further addresses bioethical considerations and regulatory frameworks necessary to ensure responsible genome engineering as a milestone, as well as the implementation of safeguards against misuse, particularly in pathogenic microorganisms. Furthermore, the convergence of standardised experimental data, specialised microbial datasets, and advanced AI architectures is paving the way to transform microbial biotechnology by accelerating metabolic engineering and synthetic biology applications. Full article

Aconitum soongaricum, a medicinal plant endemic to the Tianshan Mountains in Xinjiang, China, produces numerous natural compounds with potential medicinal value. Mitochondria function as energy hubs and play critical roles in plant development and stress adaptation; thus, their genomic composition underpins biological functions. Here, we assembled the complete mitochondrial genome of A. soongaricum using next- and third-generation sequencing data and performed comparative analyses with related species. The mitochondrial genome exhibited a typical circular structure of 487,849 bp with a GC content of 46.80%. A total of 77 genes were annotated, including 41 protein-coding genes (PCGs), three rRNAs, 31 tRNAs, and two pseudogenes. The genome showed a strong A/U bias at the third codon position and displayed C-to-U RNA editing transitions, whereas no U-to-C transitions were estimated. Maximum-likelihood phylogenetic analysis supported a close relationship among A. soongaricum, A. carmichaelii, and A. kusnezoffii, confirming the utility of mitochondrial genomes for genetic relationship inference in genus Aconitum. Divergence time estimation placed the differentiation of A. soongaricum from the other two species at approximately 4.19 million years ago (Mya). Additionally, we evaluated the expression levels of NADH dehydrogenase (nad) genes across different tissues and under drought stress using real-time PCR, revealing diverse expression patterns. Collectively, this study provides a foundation for future investigations into the genetic mechanisms underlying evolution, energy metabolism, and environmental adaptation in A. soongaricum. Full article

Early flowering-related factors play pivotal roles in coordinating plant growth and reproductive development. In this study, we investigated the biological function of early flowering gene (ELF) in Arabidopsis thaliana using CRISPR/Cas9-mediated genome editing and construction of overexpression approaches. Two independent ELF overexpression (OE-ELF) and genome-edited (ge-elf) lines were generated and systemically analyzed. ELF overexpression significantly enhanced early seedling performance, increasing germination rate and seedling fresh weight by up to 8.7%, while genome-edited lines exhibited a marked reduction. Root growth was strongly promoted in OE-ELF plants, with root length increase of 85% and 75%, whereas ge-elf lines showed a reduction of up to 48%. At later developmental stages, OE-ELF plants displayed enhanced vegetative growth, including increased leaf length (32%), leaf area (91%), and accelerated flowering (21% earlier than wild type). In contrast, ge-elf delayed flowering by up to 25% and resulted in compact plant architecture. Reproductive development was severely compromised in ge-elf plants, which exhibited malformed inflorescences, reduced pollen germination, shortened silique (45%), and a drastic decrease in seed number per silique (70%). Conversely, OE-ELF plants showed increased silique number and seed per silique. Molecular analysis revealed that ELF positively regulates key flowering-related genes, including FLC, SOC1, AP1, and LFY, which correlated strongly with growth and reproductive traits. Our results demonstrate that ELF functions as a central regulator integrating vegetative growth, floral development, male fertility, and seed production in Arabidopsis thaliana. Full article

Abiotic stresses, including drought, salinity, alkalinity, temperature extremes, flooding, heavy metals, and emerging pollutants, challenge plant growth and productivity by disturbing water relations, ion balance, redox homeostasis, membrane stability, energy metabolism, and developmental progression. Although substantial progress has been made in the identification of stress-responsive hormones, second messengers, kinases, transcription factors, transporters, and metabolic regulators, plant stress adaptation cannot be fully explained by linear signaling cascades or single tolerance genes. A major unresolved question is how early molecular events are reorganized into coordinated physiological and developmental outputs that support survival, recovery, and productivity. In this review, we propose an intermolecular interaction-driven adaptive remodeling framework for plant abiotic stress responses. This framework emphasizes that stress tolerance emerges from dynamic changes in receptor–ligand recognition, protein–protein interactions, calcium decoding, redox-sensitive modification, phosphorylation networks, transcriptional regulation, chromatin-associated control, and metabolite-mediated feedback. We further emphasize ROS as integrative redox switches that connect stress sensing, defense activation, senescence-related transitions, and recovery, and chromatin-associated mechanisms as regulators that may stabilize primed or memory-like adaptive states. We discuss how these interaction networks converge on core signaling hubs, including abscisic acid, reactive oxygen species, Ca²⁺, and kinase/phosphatase systems, and how they remodel stomatal behavior, root architecture, ion and pH homeostasis, redox buffering, metabolism, development, and reproductive resilience. We further highlight how natural variation, multi-omics, genome editing, high-throughput phenotyping, and field validation can translate interaction-centered stress biology into crop resilience. This perspective provides a conceptual bridge between molecular stress perception, network behavior, physiological adaptation, and climate-resilient agriculture. Full article

The CRISPR/Cas9 system has been widely used for gene editing in various species; however, mosaicism remains a significant challenge. This study aimed to improve gene editing efficiency and reduce mosaicism in porcine embryos by exploring double electroporation pre- and post-in vitro fertilization combined with zona pellucida (ZP) removal. We evaluated the effects of these treatments on the development and mutation rates of oocytes/zygotes edited with guide RNAs (gRNAs) targeting GGTA1, CMAH, or B4GALNT2 genes. Double electroporation significantly increased the total and biallelic mutation rates in ZP-intact zygotes but not in ZP-free zygotes edited using GGTA1-targeted gRNAs. All blastocysts from ZP-free zygotes exhibited biallelic mutations following double electroporation. For the CMAH gene, all blastocysts exhibited mutations (biallelic mutations ≥ 80%); however, double electroporation and ZP removal did not affect their mutation rates or efficiency. For the B4GALNT2 gene, double electroporation significantly increased total mutation rates in ZP-intact zygotes, whereas all blastocysts from ZP-free zygotes showed biallelic mutation. These findings suggest that double electroporation, particularly with ZP removal, may enhance gene-editing efficiency, reduce mosaicism and improve the success of genetic modifications. Full article

Endogenous gene tagging using CRISPR has changed the understanding of the role played by different proteins due to the ability to track and study proteins in their natural state. With CRISPR-based gene tagging, it is possible to insert fluorescent, luminescent, epitope, affinity, and proximity labels into the target protein at its endogenous genomic location without affecting its physiological expression and dynamics. Here, we discuss the DNA-repair mechanisms employed in endogenous gene tagging, including homology-dependent repair, NHEJ-based integration, and alternative approaches that can be used with challenging cell types. Key aspects of efficient CRISPR tagging experiments are also described. Additionally, we review recent advances in the increasing array of protein tag technologies, including fluorescent proteins, split-reporter technologies, NanoLuc/HiBiT, peptide epitopes, and proximity biotinylation enzymes. Lastly, we review the scalability of endogenous tagging approaches using multiplex editing, atlas-scale proteome tagging, iPSC-based disease modeling, and drug discovery platforms for assessing target engagement, protein degradation, phenotype screening, and mechanism of action of compounds. Although difficult in primary and pluripotent cells, new methods based on avoiding double-strand breaks, such as prime editing, PASTE, and CRISPR associated transposases, will drive the future expansion of endogenous tagging approaches. Such developments firmly set up CRISPR gene tagging as a fundamental technology in quantitative cell biology and translational pharmacology. Full article

Cyanobacteria are increasingly recognised as photosynthetic chassis for sustainable metabolic engineering because oxygenic photosynthesis generates ATP and NADPH via the photosynthetic electron transport chain, which drive CO₂ fixation through the Calvin–Benson–Bassham cycle into carbon intermediates that can be redirected toward engineered heterologous pathways. Their genetic tractability, CO₂-fixing capacity, ecological adaptability, and relatively simple cellular organisation make them attractive platforms for developing low-carbon biotechnological processes. This review explores recent progress in engineering cyanobacteria for heterologous pathway construction, critically evaluating genetic tools including transformation methods, genome integration strategies, promoter systems, and CRISPR-based editing, with specific emphasis on challenges of direct relevance to phototrophic chassis: host–pathway metabolic compatibility, precursor supply, cofactor balancing between photosynthetic output and heterologous pathway demand, and achieving genetic stability in polyploid cyanobacterial genomes. The review also addresses key limitations with mechanistic context: metabolic burden from multi-gene pathway expression reduces growth rate and selects against producing cells; polyploidy delays complete chromosomal segregation of engineered constructs; slow photoautotrophic growth constrains volumetric productivity; native regulatory networks resist carbon flux redirection; and cultivation constraints—including light attenuation in dense cultures and mismatches between photosynthetic ATP/NADPH supply and heterologous pathway demand—further limit achievable yields. Full article

Low-temperature-induced fertility restoration in thermo-sensitive genic male sterile (TGMS) lines severely impairs hybrid seed purity, which is a major bottleneck for two-line hybrid rice production. Most commercial TGMS lines rely on the single tms5 locus, leading to high climatic vulnerability. In this study, we developed a dual-locus strategy by target genome editing of TMS5 and MS1 in indica rice GH89. Adenine base editing at the MS1 locus exhibited a high editing efficiency of 93.5%. Transgene-free homozygous single mutants (GH89-tms5 and GH89-MS1) and double mutant (GH89-tms5 + MS1) were generated for phenotypic analysis. The double mutant GH89-tms5 + MS1 remained completely sterile for 5 and 10 days under controlled low temperature (23.5 °C), with only minimal fertility restoration after 15 days. In the field, it maintained complete sterility for 84 consecutive days and was fully insensitive to short-term low temperature fluctuations, outperforming single mutants and commercial control Y58S. Moreover, the double mutant retained most key yield-related agronomic traits of the wild type with only minor variations. This dual mutation forms a “double-lock” fertility regulatory system, significantly increasing the low-temperature duration threshold for fertility restoration. The GH89-tms5 + MS1 line exhibits promising potential for future rice breeding applications. Full article

Extreme climatic conditions often intensify abiotic stress factors (such as drought, salinity, heat stress, ultraviolet radiation, and soil degradation), and are increasingly limiting crop productivity and threatening global food security. Secondary metabolites (SMs), traditionally viewed as defense compounds, are now recognized as key regulators of plant adaptation to environmental stress. This review synthesizes recent advances in understanding the role of SMs as biochemical targets for improving crop resilience to climate extremes. By integrating evidence from multi-omics studies, artificial-intelligence-driven analyses, and functional genomics, we examine how stress-specific metabolic signatures and regulatory networks can be exploited for crop improvement. We further discuss the application of genome editing, synthetic biology, and metabolomics-assisted breeding to modulate the SM pathways to enhance stress tolerance. Selected case studies highlight the contribution of flavonoids, alkaloids, and terpenoids to stress adaptation in major and underutilized crops grown under salinity, drought, and low-temperature conditions. Despite significant progress, challenges remain, including metabolic trade-offs between stress tolerance and yield, regulatory constraints, and public acceptance of genetically engineered crops. By linking molecular mechanisms with applied strategies, this review provides a conceptual framework for leveraging secondary metabolism in climate-resilient agriculture and identifies key gaps to guide future research and innovation. Full article

Background/Objectives: Immune checkpoints are critical regulatory pathways that maintain peripheral tolerance and prevent autoimmunity. Among these, the programmed death-1/programmed death-ligand 1 (PD-1/PD-L1) axis serves as a major inhibitory pathway that terminates T cell responses. While protein-based checkpoint blockade (ICB) targeting this axis has revolutionized clinical cancer therapy, its clinical efficacy is frequently limited by low response rates, immune-related adverse events (irAEs), and the emergence of adaptive resistance. To break through these bottlenecks, genetic interruption has emerged as a high-precision alternative to modulate the PD-1/PD-L1 pathway at the nucleotide level. Methods: A comprehensive systematic review of literature was performed across major databases (PubMed, Web of Science), with a focus on high quality studies published up to 2026. Results: Direct genomic disruption via CRISPR/Cas9 and post-transcriptional silencing through RNA interference can effectively neutralize inhibitory signaling at its source. Recent advances demonstrate that targeting upstream regulatory nodes—including metabolic checkpoints (e.g., lactate metabolism) and biophysical mechanisms (e.g., liquid–liquid phase separation)—provides superior transcriptional control over PD-L1. Furthermore, engineering CAR-T cells with multiplex gene editing (e.g., TCR/B2M/PD-1 knockout) or localized scFv secretion significantly enhances antitumor potency while reducing systemic toxicity. Innovations in organ-targeted lipid nanoparticles and stimuli-responsive biomimetic carriers further address the delivery barriers in solid tumors. Conclusions: Gene therapy provides a high-precision platform for PD-1/PD-L1 modulation, offering a viable strategy to overcome adaptive resistance. Future clinical application depends on the refinement of safer editing tools, such as base editing, and the standardization of intelligent delivery systems to ensure controllable and scalable cancer immunotherapy. Full article

Mitochondrial DNA (mtDNA) mutations are associated with a diverse spectrum of diseases and pose a significant threat to human health. Despite their importance as therapeutic targets, the unique structural and electrochemical properties of mitochondria—most notably the impermeable inner mitochondrial membrane and the high membrane potential—present formidable challenges for the targeted delivery of therapeutic agents. Currently, there are no approved curative treatments for patients harboring pathogenic mtDNA mutations. In this review, we discuss recent advancements in gene therapy for mitochondrial genome-related disorders, with a particular focus on allotopic expression of mtDNA-encoded genes and mitochondrial genome editing technologies. We conclude that allotopic expression currently stands as the most promising approach for near-term clinical implementation. But we also pay great attention to programmable nucleases and base editors utilizing RNA-independent DNA recognition which are evolving with remarkable speed. Full article