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: 26 245
Страница 892 из 1000-
RNA-guided gene editing of the murine gammaherpesvirus 68 genome reduces infectious virus production.
Epstein-Barr virus (EBV) and Kaposi sarcoma herpesvirus (KSHV) are cancer-causing viruses that establish lifelong infections in humans. Gene editing using the Cas9-guideRNA (gRNA) CRISPR system has been applied to decrease the latent load of EBV in human Burkitt lymphoma cells. Validating the efficacy of Cas9-gRNA system in eradicating infection in vivo without off-target effects to the host genome will require animal model systems. To this end, we evaluated a series of gRNAs against individual genes and functional genomic elements of murine gammaherpesvirus 68 (MHV68) that are both conserved with KSHV and important for the establishment of latency or reactivation from latency in the host. gRNA sequences against ORF50, ORF72 and ORF73 led to insertion, deletion and substitution mutations in these target regions of the genome in cell culture. Murine NIH3T3 fibroblast cells that stably express Cas9 and gRNAs to ORF50 were most resistant to replication upon de novo infection. Latent murine A20 B cell lines that stably express Cas9 and gRNAs against MHV68 were reduced in their reactivation by approximately 50%, regardless of the viral gene target. Lastly, co-transfection of HEK293T cells with the vector expressing the Cas9-MHV68 gRNA components along with the viral genome provided a rapid read-out of gene editing and biological impact. Combinatorial, multiplex MHV68 gRNA transfections in HEK293T cells led to near complete ablation of infectious particle production. Our findings indicate that Cas9-gRNA editing of the murine gammaherpesvirus genome has a deleterious impact on productive replication in three independent infection systems.
-
Development of Locus-Directed Editing of the Epigenome from Basic Mechanistic Engineering to First Clinical Applications.
The introduction of CRISPR/Cas systems has resulted in a strong impulse for the field of gene-targeted epigenome/epigenetic reprogramming (EpiEditing), where EpiEditors consisting of a DNA binding part for targeting and an enzymatic part for rewriting of chromatin modifications are applied in cells to alter chromatin modifications at targeted genome loci in a directed manner. Pioneering studies preceding this era indicated causal relationships of chromatin marks instructing gene expression. The accumulating evidence of chromatin reprogramming of a given genomic locus resulting in gene expression changes opened the field for mainstream applications of this technology in basic and clinical research. The growing knowledge on chromatin biology and application of EpiEditing tools, however, also revealed a lack of predictability of the efficiency of EpiEditing in some cases. In this perspective, the dependence of critical parameters such as specificity, effectivity, and sustainability of EpiEditing on experimental settings and conditions including the expression levels and expression times of the EpiEditors, their chromatin binding affinity and specificity, and the crosstalk between EpiEditors and cellular epigenome modifiers are discussed. These considerations highlight the intimate connection between the outcome of epigenome reprogramming and the details of the technical approaches toward EpiEditing, which are the main topic of this volume of Methods in Molecular Biology. Once established in a fully functional "plug-and-play" mode, EpiEditing will allow to better understand gene expression control and to translate such knowledge into therapeutic tools. These expectations are beginning to be met as shown by various in vivo EpiEditing applications published in recent years, several companies aiming to exploit the therapeutic power of EpiEditing and the first clinical trial initiated.
-
Current approaches in CRISPR-Cas system for metabolic disorder.
A new era in genomic medicine has been brought by the development of CRISPR-Cas technology, which presents hitherto unheard-of possibilities for the treatment of metabolic illnesses. The treatment approaches used in CRISPR/Cas9-mediated gene therapy, emphasize distribution techniques such as viral vectors and their use in preclinical models of metabolic diseases like hypercholesterolemia, glycogen storage diseases, and phenylketonuria. The relevance of high-throughput CRISPR screens for target identification in discovering new genes and pathways associated with metabolic dysfunctions is an important aspect of the discovery of new approaches. With cutting-edge options for genetic correction and cellular regeneration, the combination of CRISPR-Cas technology with stem cell therapy has opened new avenues for the treatment of metabolic illnesses. The integration of stem cell therapy and CRISPR-Cas technology is an important advance in the treatment of metabolic diseases, which are difficult to treat because of their intricate genetic foundations. This chapter addresses the most recent developments in the application of stem cell therapy and CRISPR-Cas systems to treat a variety of metabolic disorders, providing fresh hope for effective and maybe curative therapies. This chapter examines techniques and developments that have been made recently to address a variety of metabolic disorders using CRISPR-Cas systems. Our chapter focuses on the foundational workings of CRISPR-Cas technology and its potential uses in gene editing, gene knockout, and activation/repression-based gene modification.
-
Allele-specific genome targeting in the development of precision medicine.
The CRISPR-based genome editing holds immense potential to fix disease-causing mutations, however, must also handle substantial natural genetic variations between individuals. Previous studies have shown that mismatches between the single guide RNA (sgRNA) and genomic DNA may negatively impact sgRNA efficiencies and lead to imprecise specificity prediction. Hence, the genetic variations bring about a great challenge for designing platinum sgRNAs in large human populations. However, they also provide a promising entry for designing allele-specific sgRNAs for the treatment of each individual. The CRISPR system is rather specific, with the potential ability to discriminate between similar alleles, even based on a single nucleotide difference. Genetic variants contribute to the discrimination capabilities, once they generate a novel protospacer adjacent motif (PAM) site or locate in the seed region near an available PAM. Therefore, it can be leveraged to establish allele-specific targeting in numerous dominant human disorders, by selectively ablating the deleterious alleles. So far, allele-specific CRISPR has been increasingly implemented not only in treating dominantly inherited diseases, but also in research areas such as genome imprinting, haploinsufficiency, spatiotemporal loci imaging and immunocompatible manipulations. In this review, we will describe the working principles of allele-specific genome manipulations by virtue of expanding engineering tools of CRISPR. And then we will review new advances in the versatile applications of allele-specific CRISPR targeting in treating human genetic diseases, as well as in a series of other interesting research areas. Lastly, we will discuss their potential therapeutic utilities and considerations in the era of precision medicine.
-
CRISPR-Cas guides the future of genetic engineering.
The diversity, modularity, and efficacy of CRISPR-Cas systems are driving a biotechnological revolution. RNA-guided Cas enzymes have been adopted as tools to manipulate the genomes of cultured cells, animals, and plants, accelerating the pace of fundamental research and enabling clinical and agricultural breakthroughs. We describe the basic mechanisms that set the CRISPR-Cas toolkit apart from other programmable gene-editing technologies, highlighting the diverse and naturally evolved systems now functionalized as biotechnologies. We discuss the rapidly evolving landscape of CRISPR-Cas applications, from gene editing to transcriptional regulation, imaging, and diagnostics. Continuing functional dissection and an expanding landscape of applications position CRISPR-Cas tools at the cutting edge of nucleic acid manipulation that is rewriting biology.
-
[Identify Myeloid Differentiation-Related MiRNAs Response to ATRA Induction by RNA Sequencing and CRISPR/Cas9 Gene Editing].
OBJECTIVE: To identify differentiation related miRNA and evaluate roles of miRNA during ATRA induced myeloid differentiation. METHODS: The small RNA sequencing was used to analyze differential expressed miRNAs in ATRA induced NB4 cells. Then the several up or down-regulated miRNA were selected as the research candidates. SgRNAs targeting the genome of each miRNA were designed and NB4 cells with inducible expression of Cas9 protein were generated. After transduced sgRNA into NB4/Cas9 cells, the mutation level by PCR and surveyor assay were evaluated. The cell differentiation level was investigated by surface CD11b expression via flow cytometry. RESULTS: A total of 410 mature miRNAs which expressed in NB4 cells were detected out after treated by ATRA, 74 miRNAs were up-regulated and 55 were down-regulated miRNAs with DNA cleavage generated by CRISPR/Cas9 was assayed directly by PCR or surveyor assay, quantitative PCR showed that the expression of miRNA was downregulated, which evaluated that gene edition successfully inhibitied the expression of mature miRNA. MiR-223 knockout showed the myeloid differentation of NB4 significantly inhibitied, while miRNA-155 knockout showed the myeloid differentation of NB4 cells significantly increased. CONCLUSION: CRISPR/Cas9 is a powerful tool for gene editing and can lead to miRNA knockout. Knockouts of miR-223 and miR-155 have shown a differentiation-related phenotype, and the potential mechanism is the integrative regulation of target genes.
-
CRISPR/Cas genome editing perspectives for barley breeding.
The CRISPR/Cas9 technology shows potential to improve crop breeding efficiency and antiviral defense. The interest in DNA editing in crops has grown due to the possibility of increasing the resistance of different plants to many viruses. Our aim was to create an elite disease-resistant local barley cultivar using CRISPR/Cas9 biotechnology. For this purpose, we used CRISPR/Cas 9-eIF4E with the eukaryotic translation initiation factor 4E (eIF4E) barley gene to edit the genomes of five local Kazakhstan barley cultivars. After identifying the single guide RNA (sgRNA) target sequences, they were synthesized and cloned into the CRISPR-plant vector before being introduced into barley cells via our own patented Agrobacterium germ-line transformation technique. Barley plants eIF4E-modified were successfully obtained and were resistant to virus infection. Based on our research, the CRISPR/Cas9 system for plant genome editing could be a prospect for applying this breakthrough biotechnology in barley breeding.
-
Driving the wedge: Understanding an improved Cas9 to better engineer others.
In their recent structural work, Eggers et al.1 rationalize how key mutations in the WED domain of the compact and thermostable Geobacillus stearothermophilus Cas9 bolster its editing efficiency in mammalian cells, and they use these insights to rationally improve another Cas9.
-
Knockout of Glycosyltransferases in Nicotiana benthamiana by Genome Editing to Improve Glycosylation of Plant-Produced Proteins.
Plants are excellent production hosts for the in vivo synthesis of complex glycosylated proteins such as antibodies. The plant N-glycosylation machinery is largely similar to that found in humans and other mammalian organisms, which is an advantage in comparison to microbial production systems in particular. However, there are some differences in the identity and chemical linkage of the sugars that plants and mammals use to build their N-glycans. These differences can affect important properties of glycosylated proteins produced recombinantly in plants. Here we describe the complete procedure of multiplex targeted gene knockout with CRISPR/Cas9 in Nicotiana benthamiana in order to eliminate the undesirable sugars α-1,3-fucose and β-1,2-xylose from the plant N-glycans. The workflow includes target gene identification, guide RNA design and testing, plant transformation, and the analysis of the regenerated transgenic plants by Sanger sequencing, immunoblot, and mass-spectrometric analysis of recombinant and endogenous proteins.
-
Advances in protoplast transfection promote efficient CRISPR/Cas9-mediated genome editing in tetraploid potato.
An efficient method of DNA-free gene-editing in potato protoplasts was developed using linearized DNA fragments, UBIQUITIN10 promoters of several plant species, kanamycin selection, and transient overexpression of the BABYBOOM transcription factor. Plant protoplasts represent a reliable experimental system for the genetic manipulation of desired traits using gene editing. Nevertheless, the selection and regeneration of mutated protoplasts are challenging and subsequent recovery of successfully edited plants is a significant bottleneck in advanced plant breeding technologies. In an effort to alleviate the obstacles related to protoplasts' transgene expression and protoplasts' regeneration, a new method was developed. In so doing, it was shown that linearized DNA could efficiently transfect potato protoplasts and that UBIQUITIN10 promoters from various plants could direct transgene expression in an effective manner. Also, the inhibitory concentration of kanamycin was standardized for transfected protoplasts, and the NEOMYCIN PHOSPHOTRANSFERASE2 (NPT2) gene could be used as a potent selection marker for the enrichment of transfected protoplasts. Furthermore, transient expression of the BABYBOOM (BBM) transcription factor promoted the regeneration of protoplast-derived calli. Together, these methods significantly increased the selection for protoplasts that displayed high transgene expression, and thereby significantly increased the rate of gene editing events in protoplast-derived calli to 95%. The method developed in this study facilitated gene-editing in tetraploid potato plants and opened the way to sophisticated genetic manipulation in polyploid organisms.
PubMed отдаёт не больше 10 000 первых результатов — уточните запрос, чтобы увидеть остальные.