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Application of Genome Editing in Tomato Breeding: Mechanisms, Advances, and Prospects.
Plants regularly face the changing climatic conditions that cause biotic and abiotic stress responses. The abiotic stresses are the primary constraints affecting crop yield and nutritional quality in many crop plants. The advances in genome sequencing and high-throughput approaches have enabled the researchers to use genome editing tools for the functional characterization of many genes useful for crop improvement. The present review focuses on the genome editing tools for improving many traits such as disease resistance, abiotic stress tolerance, yield, quality, and nutritional aspects of tomato. Many candidate genes conferring tolerance to abiotic stresses such as heat, cold, drought, and salinity stress have been successfully manipulated by gene modification and editing techniques such as RNA interference, insertional mutagenesis, and clustered regularly interspaced short palindromic repeat (CRISPR/Cas9). In this regard, the genome editing tools such as CRISPR/Cas9, which is a fast and efficient technology that can be exploited to explore the genetic resources for the improvement of tomato and other crop plants in terms of stress tolerance and nutritional quality. The review presents examples of gene editing responsible for conferring both biotic and abiotic stresses in tomato simultaneously. The literature on using this powerful technology to improve fruit quality, yield, and nutritional aspects in tomato is highlighted. Finally, the prospects and challenges of genome editing, public and political acceptance in tomato are discussed.
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Haplotype editing with CRISPR-Cas9 as a therapeutic approach for dominant-negative missense mutations in NEFL.
Inactivation of disease alleles by allele-specific editing is a promising approach to treat dominant-negative genetic disorders, provided the causative gene is haplosufficient. We previously edited a dominant NEFL missense mutation causing Charcot-Marie-Tooth type 2E (CMT2E) with inactivating frameshifts and rescued disease-relevant phenotypes in induced pluripotent stem cell (iPSC)-derived motor neurons. However, a multitude of different NEFL missense mutations cause CMT2E. Here, we addressed this challenge by targeting common single-nucleotide polymorphisms in cis with NEFL disease mutations for gene excision. We validated this haplotype editing approach in two iPSC lines with different missense mutations and demonstrated phenotypic rescue in iPSC-motor neurons. Surprisingly, our analysis revealed that gene inversion, a frequent by-product of excision editing, failed to reliably disrupt mutant allele expression. We deployed novel molecular assays to optimize our approach and achieve therapeutic levels of editing in immature iPSC-motor neurons. Finally, population genetics analysis demonstrated the power of haplotype editing to enable therapeutic development for the greatest number of patients. Our data serve as an important case study for many dominant genetic disorders amenable to this approach.
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Utilization of the CRISPR/Cas9 system for the efficient production of mutant mice using crRNA/tracrRNA with Cas9 nickase and FokI-dCas9.
The CRISPR/Cas9 system is a powerful genome editing tool for the production of genetically modified animals. To produce mutant mice, chimeric single-guide RNA (sgRNA) is cloned in a plasmid vector and a mixture of sgRNA and Cas9 are microinjected into the fertilized eggs. An issue associated with gene manipulation using the CRISPR/Cas9 system is that there can be off-target effects. To simplify the production of mutant mice with low risks of off-target effects caused by the CRISPR/Cas9 system, we demonstrated that genetically modified mice can be efficiently obtained using chemically synthesized CRISPR RNA (crRNA), trans-activating crRNA (tracrRNA), and modified Cas9s, such as the nickase version and FokI-fused catalytically inactive Cas9, by microinjection into fertilized eggs. Using this method, it is no longer necessary to clone sgRNA into a plasmid vector, and this enables high-throughput production of mutant mice.
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Optimization Strategy for Generating Gene-edited Tibet Minipigs by Synchronized Oestrus and Cytoplasmic Microinjection.
The Tibet minipig is a rare highland pig breed worldwide and has many applications in biomedical and agricultural research. However, Tibet minipigs are not like domesticated pigs in that their ovulation number is low, which is unfavourable for the collection of zygotes. Partly for this reason, few studies have reported the successful generation of genetically modified Tibet minipigs by zygote injection. To address this issue, we described an efficient way to generate gene-edited Tibet minipigs, the major elements of which include the utilization of synchronized oestrus instead of superovulation to obtain zygotes, optimization of the preparation strategy, and co-injection of clustered regularly interspaced short palindromic repeat sequences associated protein 9 (Cas9) mRNA and single-guide RNAs (sgRNAs) into the cytoplasm of zygotes. We successfully obtained allelic TYR gene knockout (TYR-/-) Tibet minipigs with a typical albino phenotype (i.e., red-coloured eyes with light pink-tinted irises and no pigmentation in the skin and hair) as well as TYR-/-IL2RG-/- and TYR-/-RAG1-/- Tibet minipigs with typical phenotypes of albinism and immunodeficiency, which was characterized by thymic atrophy and abnormal immunocyte proportions. The overall gene editing efficiency was 75% for the TYR single gene knockout, while for TYR-IL2RG and TYR-RAG1 dual gene editing, the values were 25% and 75%, respectively. No detectable off-target mutations were observed. By intercrossing F0 generation minipigs, targeted genetic mutations can also be transmitted to gene-edited minipigs' offspring through germ line transmission. This study is a valuable exploration for the efficient generation of gene-edited Tibet minipigs with medical research value in the future.
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Spatiotemporal Delivery of CRISPR/Cas9 Genome Editing Machinery Using Stimuli-Responsive Vehicles.
Recent innovations in genome editing have enabled the precise manipulation of the genetic information of mammalians, and benefitted the development of next-generation gene therapy. Despite these advances, several barriers to the clinical translation of genome editing remain, including the intracellular delivery of genome editing machinery, and the risk of off-target editing effect. Here, we review the recent advance of spatiotemporal delivery of CRISPR/Cas9 genome editing machinery, which is composed of programmable Cas9 nuclease and a single-guide RNA (sgRNA) using stimuli-responsive nanoparticles. We discuss the specific chemistries that have been used for controlled Cas9/sgRNA delivery and intracellular release in the presence of endogenous or external signals. These methodologies can leverage biological signals found locally within disease cells, or exogenous signals administrated with spatiotemporal control, through which an improved genome editing could be achieved. We also discuss the future in exploiting these approaches for fundamental biomedical applications and therapeutic genome editing.
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Revolutionising cancer intervention: the repercussions of CAR-T cell therapy on modern oncology practices.
Chimeric Antigen Receptor T-cell (CAR-T) therapy represents a groundbreaking advance in oncology, leveraging patient-specific immune cells to target malignant tumours precisely. By equipping T cells with synthetic receptors, CAR-T therapy achieves remarkable antitumor effects and offers hope for durable cancer control. However, several limitations persist, including antigen scarcity, immunosuppressive tumour microenvironments, and T-cell exhaustion. CRISPR-Cas9 gene editing has enhanced CAR-T potency by knocking out immune checkpoints (PD-1, CTLA-4) and improving persistence, while RNA interference (RNAi) silences immune-evasion genes (e.g. SOCS1). Nanozyme-based delivery systems enable precise CRISPR-Cas9 delivery (> 70% editing efficiency) and tumour targeting, overcoming instability and off-target effects. Innovations like SUPRA CARs, armoured CAR-T cells (e.g. IL-12/IL-21-secreting TRUCKs), and dual checkpoint inhibition synergize to reprogram the tumour microenvironment, reducing relapse by 40% in trials. Despite progress, high costs, manufacturing hurdles, and ethical concerns (e.g. germline editing risks) remain critical barriers. Emerging solutions include universal off-the-shelf CAR-Ts, hybrid nano-CRISPR systems, and AI-driven design, paving the way for scalable, personalised immunotherapy. This review highlights breakthroughs in CRISPR, RNAi, and nanotechnology, underscoring CAR-T therapy's transformative potential while addressing translational challenges for broader clinical adoption.
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A CRISPR-Cas9 System for Genome Editing of Fusarium proliferatum.
Fusarium proliferatum causes diverse diseases of many economically important plants. The fungus produces several mycotoxins of which the fumonisins are the most toxic. Currently, deletion of key genes for mycotoxin biosynthesis is a laborious and time-consuming procedure. We developed a novel CRISPR/Cas9-based genome-editing tool for the direct delivery of preassembled Cas9 ribonucleoproteins into protoplasts of F. proliferatum. Our CRISPR-Cas9 system couples a site-specific double-strand DNA break mediated by two Cas9 ribonucleoproteins with microhomology recombination requiring only 50-bp regions flanking the target gene. This system reduces the risk of off-target mutations and minimizes the risk of altering any gene adjacent to the target region. We used this tool to delete a polyketide synthase gene (FUM1) required for fumonisin biosynthesis. The mutants generated are no longer able to produce fumonisins, confirming the key role of FUM1 in fumonisin biosynthesis. Our CRISPR-Cas9 system is an important new tool for genetic studies of Fusarium.
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Repair of CRISPR-guided RNA breaks enables site-specific RNA excision in human cells.
Genome editing with CRISPR RNA-guided endonucleases generates DNA breaks that are resolved by cellular DNA repair machinery. However, analogous methods to manipulate RNA remain unavailable. We show that site-specific RNA breaks generated with type-III CRISPR complexes are repaired in human cells and that this repair can be used for programmable deletions in human transcripts to restore gene function. Collectively, this work establishes a technology for precise RNA manipulation with potential therapeutic applications.
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What we can learn from the first personalized CRISPR-treated baby to tackle genetic brain disorders.
The landmark report on personalized CRISPR genome editing to treat an infant (baby KJ) with a life-threatening liver disease sparked widespread attention,1 ushering in a new era of precision genetic intervention. This piece discusses the key challenges and opportunities in translating this milestone into treatments for genetic brain disorders.
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Control of gene editing by manipulation of DNA repair mechanisms.
DNA double-strand breaks (DSBs) are produced intentionally by RNA-guided nucleases to achieve genome editing through DSB repair. These breaks are repaired by one of two main repair pathways, classic non-homologous end joining (c-NHEJ) and homology-directed repair (HDR), the latter being restricted to the S/G2 phases of the cell cycle and notably less frequent. Precise genome editing applications rely on HDR, with the abundant c-NHEJ formed mutations presenting a barrier to achieving high rates of precise sequence modifications. Here, we give an overview of HDR- and c-NHEJ-mediated DSB repair in gene editing and summarize the current efforts to promote HDR over c-NHEJ.
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