Браузер не даёт сохранять данные (например, приватный режим): закладки пропадут после закрытия вкладки.
Поиск статей PubMed
Ключевые слова, автор, журнал, годы — результаты из PubMed, аннотации с переводом на русский, закладки в браузере.
Найдено: 26 182
Страница 988 из 1000-
Delivery of an Artificial Transcription Regulator dCas9-VPR by Extracellular Vesicles for Therapeutic Gene Activation.
The CRISPR/Cas system has been developed as a potent tool for genome engineering and transcription regulation. However, the efficiency of the delivery of the system into cells, particularly for therapeutic in vivo applications, remains a major bottleneck. Extracellular vesicles (EVs), released by eukaryotic cells, can mediate the transfer of various molecules, including nucleic acids and proteins. We show the packaging and delivery of the CRISPR/Cas system via EVs to the target cells, combining the advantages of both technological platforms. A genome editing with designed extracellular vesicles (GEDEX) system generated by the producer cells can transfer the designed transcriptional regulator dCas9-VPR complexed with appropriate targeting gRNAs enabling activation of gene transcription. We show functional delivery in mammalian cells as well in the animals. The therapeutic efficiency of in vivo delivery of dCas9-VPR/sgRNA GEDEX is demonstrated in a mouse model of liver damage counteracted by upregulation of the endogenous hepatocyte growth factor, demonstrating the potential for therapeutic applications.
-
Creating a zero amylose barley with high soluble sugar content by genome editing.
Amylose biosynthesis is strictly associated with granule-bound starch synthase I (GBSSI) encoded by the Waxy gene. Mutagenesis of single bases in the Waxy gene, which induced by CRISPR/Cas9 genome editing, caused absence of intact GBSSI protein in grain of the edited line. The amylose and amylopectin contents of waxy mutants were zero and 31.73%, while those in the wild type were 33.50% and 39.00%, respectively. The absence of GBSSI protein led to increase in soluble sugar content to 37.30% compared with only 10.0% in the wild type. Sucrose and β-glucan, were 39.16% and 35.40% higher in waxy mutants than in the wild type, respectively. Transcriptome analysis identified differences between the wild type and waxy mutants that could partly explain the reduction in amylose and amylopectin contents and the increase in soluble sugar, sucrose and β-glucan contents. This waxy flour, which showed lower final viscosity and setback, and higher breakdown, could provide more option for food processing.
-
CRISPR/Cas9 Delivery Using Extracellular Vesicles.
Extracellular vesicles (EVs), particles released from cells, have the potential to become an important in vivo delivery vehicle for CRISPR machinery. Unlike viral vectors such as Adeno-associated virus (AAV), EVs reduce risks associated with immunogenicity, long-term expression, and potential off-target effects. EVs can efficiently deliver CRISPR/Cas9 ribonucleoprotein (RNP) complexes, which provide transient, ready-to-function editing machinery with reduced off-target risk compared with plasmid DNA or mRNA delivery. RNP loading into EVs can occur without specific targeting signals, although strategies such as membrane anchoring, inducible dimerization systems, or fusion with EV-associated proteins can enhance cargo enrichment. EVs are typically produced by transfecting producer cells with plasmids encoding Cas9 and sgRNA, followed by vesicle release into culture media. Purification requires removal of cellular debris and enrichment of vesicles using methods such as ultracentrifugation, ultrafiltration, chromatography, or precipitation. As no single gold-standard purification approach exists, method selection should balance yield and purity, and characterization using vesicle markers and size distribution profiling is recommended.
-
Editing human hematopoietic stem cells: advances and challenges.
Genome editing of hematopoietic stem and progenitor cells is being developed for the treatment of several inherited disorders of the hematopoietic system. The adaptation of CRISPR-Cas9-based technologies to make precise changes to the genome, and developments in altering the specificity and efficiency, and improving the delivery of nucleases to target cells have led to several breakthroughs. Many clinical trials are ongoing, and several pre-clinical models have been reported that would allow these genetic therapies to one day offer a potential cure to patients with diseases where limited options currently exist. However, there remain several challenges with respect to establishing safety, expanding accessibility and improving the manufacturing processes of these therapeutic products. This review focuses on some of the recent advances in the field of genome editing of hematopoietic stem and progenitor cells and illustrates the ongoing challenges.
-
A Genome Editing System for Therapeutical Targeting of Stem Cells.
The CRISPR/Cas9 system can be exploited to disrupt genes or cis-regulatory elements in the genome of human hematopoietic stem cells. Here, we describe a protocol to deliver the CRISPR/Cas9 ribonucleoprotein complexes into primary human hematopoietic stem cells and to evaluate the engraftment and multilineage differentiation of edited cells in immunodeficient mice. This procedure allows the editing of a high proportion of long-term repopulating hematopoietic stem cells.
-
Germ line genome editing in clinics: the approaches, objectives and global society.
Genome editing allows for the versatile genetic modification of somatic cells, germ cells and embryos. In particular, CRISPR/Cas9 is worldwide used in biomedical research. Although the first report on Cas9-mediated gene modification in human embryos focused on the prevention of a genetic disease in offspring, it raised profound ethical and social concerns over the safety of subsequent generations and the potential misuse of genome editing for human enhancement. The present article considers germ line genome editing approaches from various clinical and ethical viewpoints and explores its objectives. The risks and benefits of the following three likely objectives are assessed: the prevention of monogenic diseases, personalized assisted reproductive technology (ART) and genetic enhancement. Although genetic enhancement should be avoided, the international regulatory landscape suggests the inevitability of this misuse at ART centers. Under these circumstances, possible regulatory responses and the potential roles of public dialogue are discussed.
-
Recent Advances in Genetic Engineering Strategies of Sinorhizobium meliloti.
Sinorhizobium meliloti is a free-living soil Gram-negative bacterium that participates in nitrogen-fixation symbiosis with several legumes. S. meliloti has the potential to be utilized for the production of high-value nutritional compounds, such as vitamin B12. Advances in gene editing tools play a vital role in the development of S. meliloti strains with enhanced characteristics for biotechnological applications. Several novel genetic engineering strategies have emerged in recent years to investigate genetic modifications in S. meliloti. This review provides a comprehensive overview of the mechanism and application of the extensively used Tn5-mediated genetic engineering strategies. Strategies based on homologous recombination and site-specific recombination were also discussed. Subsequently, the development and application of the genetic engineering strategies utilizing various CRISPR/Cas systems in S. meliloti are summarized. This review may stimulate research interest among scientists, foster studies in the application areas of S. meliloti, and serve as a reference for the utilization of genome editing tools for other Rhizobium species.
-
Cas9-Geminin and Cdt1-fused anti-CRISPR protein synergistically increase editing accuracy.
Genome editing with CRISPR-Cas9, particularly for therapeutic purposes, should be accomplished via the homology-directed repair (HDR) pathway, which exhibits greater precision than other pathways. However, one of the issues to be solved is that genome editing efficiency with HDR is generally low. A Streptococcus pyogenes Cas9 (SpyCas9) fusion with human Geminin (Cas9-Gem) reportedly increases HDR efficiency slightly. In contrast, we found that regulation of SpyCas9 activity with an anti-CRISPR protein (AcrIIA4) fused to Chromatin licensing and DNA replication factor 1 (Cdt1) significantly increases HDR efficiency and reduces off-target effects. Here, another anti-CRISPR protein, AcrIIA5, was applied, and the combined use of Cas9-Gem and Anti-CRISPR+Cdt1 showed synergistic enhancement of HDR efficiency. The method may be applicable to various anti-CRISPR/CRISPR-Cas combinations.
-
Gene targeting of dikaryotic Pleurotus ostreatus nuclei using the CRISPR/Cas9 system.
Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9)-assisted gene targeting is a promising method used in molecular breeding. We recently reported the successful introduction of this method in the monokaryotic Pleurotus ostreatus (oyster mushroom), PC9. However, considering their application in mushroom breeding, dikaryotic strains (with targeted gene mutations in both nuclei) need to be generated. This is laborious and time-consuming because a classical crossing technique is used. Herein, we report a technique that targets both nuclei of dikaryotic P. ostreatus, PC9×#64 in a transformation experiment using plasmid-based CRISPR/Cas9, with the aim of developing a method for efficient and rapid molecular breeding. As an example, we targeted strains with low basidiospore production ability through the meiosis-related genes mer3 or msh4. Four different plasmids containing expression cassettes for Cas9 and two different gRNAs targeting mer3 or msh4 were constructed and separately introduced into PC9×#64. Eight of the 38 dikaryotic transformants analyzed produced no basidiospores. Genomic PCR suggested that msh4 or mer3 mutations were introduced into both nuclei of seven out of eight strains. Thus, in this study, we demonstrated simultaneous gene targeting using our CRISPR/Cas9 system, which may be useful for the molecular breeding of cultivated agaricomycetes.
-
Engineered CRISPR-Cas9 Vector for Efficient Agrobacterium-Mediated Transformation of Phanerochaete chrysosporium.
Phanerochaete chrysosporium is a model white-rot-fungus with exceptional lignolytic activity, but its genetic manipulation has remained a major challenge due to low transformation efficiencies. Here, we report the development of an optimized Agrobacterium-mediated transformation (AMT) protocol optimized for this species. A CRISPR-Cas9 compatible binary vector was modified by replacing the mCherry cassette with a strong sGFP reporter under the constitutive 35S promoter, enabling reliable identification of transformants. Using mycelial disks as starting material and applying a sandwich selection method, we achieved stable integration of T-DNA carrying the hygromycin resistance and sGFP genes. Transformation efficiency was influenced by co-cultivation parameters, with low-temperature incubation (23°C) and the addition of acetosyringone significantly improving recovery of resistant colonies. Stable expression of sGFP was observed in transformed mycelia, confirming the reproducibility of the system. Although CRISPR-Cas9-mediated gene editing was not demonstrated in this study, our results provide proof-of-concept that a Cas9-based vector can be stably maintained in P. chrysosporium and drive heterologous gene expression. This work represents the first step toward establishing a functional genome editing platform in this ligninolytic fungus. By expanding the molecular toolbox for P. chrysosporium, the optimized transformation strategy lays the foundation for metabolic engineering aimed at lignin valorization, bioplastics, biofuels, and high-value biochemical production within a sustainable circular bioeconomy framework.
PubMed отдаёт не больше 10 000 первых результатов — уточните запрос, чтобы увидеть остальные.