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 881 из 1000-
Non-viral delivery of CRISPR-Cas9 complexes for targeted gene editing via a polymer delivery system.
Recent advances in molecular biology have led to the CRISPR revolution, but the lack of an efficient and safe delivery system into cells and tissues continues to hinder clinical translation of CRISPR approaches. Polymeric vectors offer an attractive alternative to viruses as delivery vectors due to their large packaging capacity and safety profile. In this paper, we have demonstrated the potential use of a highly branched poly(β-amino ester) polymer, HPAE-EB, to enable genomic editing via CRISPRCas9-targeted genomic excision of exon 80 in the COL7A1 gene, through a dual-guide RNA sequence system. The biophysical properties of HPAE-EB were screened in a human embryonic 293 cell line (HEK293), to elucidate optimal conditions for efficient and cytocompatible delivery of a DNA construct encoding Cas9 along with two RNA guides, obtaining 15-20% target genomic excision. When translated to human recessive dystrophic epidermolysis bullosa (RDEB) keratinocytes, transfection efficiency and targeted genomic excision dropped. However, upon delivery of CRISPR-Cas9 as a ribonucleoprotein complex, targeted genomic deletion of exon 80 was increased to over 40%. Our study provides renewed perspective for the further development of polymer delivery systems for application in the gene editing field in general, and specifically for the treatment of RDEB.
-
Enhancing the quality of staple food crops through CRISPR/Cas-mediated site-directed mutagenesis.
The enhancement of CRISPR-Cas gene editing with robust nuclease activity promotes genetic modification of desirable agronomic traits, such as resistance to pathogens, drought tolerance, nutritional value, and yield-related traits in crops. The genetic diversity of food crops has reduced tremendously over the past twelve millennia due to plant domestication. This reduction presents significant challenges for the future especially considering the risks posed by global climate change to food production. While crops with improved phenotypes have been generated through crossbreeding, mutation breeding, and transgenic breeding over the years, improving phenotypic traits through precise genetic diversification has been challenging. The challenges are broadly associated with the randomness of genetic recombination and conventional mutagenesis. This review highlights how emerging gene-editing technologies reduce the burden and time necessary for developing desired traits in plants. Our focus is to provide readers with an overview of the advances in CRISPR-Cas-based genome editing for crop improvement. The use of CRISPR-Cas systems in generating genetic diversity to enhance the quality and nutritional value of staple food crops is discussed. We also outlined recent applications of CRISPR-Cas in developing pest-resistant crops and removing unwanted traits, such as allergenicity from crops. Genome editing tools continue to evolve and present unprecedented opportunities to enhance crop germplasm via precise mutations at the desired loci of the plant genome.
-
Isolation and Analysis of a Genome-Edited Single-Hepatocyte from a Cas9 Transgenic Mouse Line.
The primary cells isolated from the freshly dissected organ are thought to be different from those cultured for a long time in vitro. For instance, hepatocytes isolated in situ from the liver, display the ability to produce albumin, cultured for about a week often tend to cease production of albumin, including loss of proliferation capability. Thus, it is difficult to perform genome editing (i.e., production of genome-edited hepatocytes by in vitro gene delivery) in such cultured cells. Furthermore, hepatic cell lines available so far do not produce albumin and they would also have lost several characteristics of native liver cells. This poses a serious disadvantage when researchers want to study gene expression profiles under specific experimental settings, for example before and after genome editing. However, this demerit can be overcome if genome-editing is performed in situ in liver and single hepatocytes (both genome-edited and wild-type) can be isolated for analysis immediately following transient gene editing. Previously, we demonstrated successful isolation of genome-edited single hepatocytes, using mice expressing systemic Cas9 transgene (called "sCAT" mouse) and by tail-vein-mediated hydrodynamics-based gene delivery of gRNA targeted to Albumin gene (Sakurai et al., Sci Rep 6:20011, 2016). Here, we describe the detailed protocols for collection and analysis of single genome-edited hepatocytes, which will be useful for many types of hepatocyte functional studies.
-
Engineering a far-red light-activated split-Cas9 system for remote-controlled genome editing of internal organs and tumors.
It is widely understood that CRISPR-Cas9 technology is revolutionary, with well-recognized issues including the potential for off-target edits and the attendant need for spatiotemporal control of editing. Here, we describe a far-red light (FRL)-activated split-Cas9 (FAST) system that can robustly induce gene editing in both mammalian cells and mice. Through light-emitting diode-based FRL illumination, the FAST system can efficiently edit genes, including nonhomologous end joining and homology-directed repair, for multiple loci in human cells. Further, we show that FAST readily achieves FRL-induced editing of internal organs in tdTomato reporter mice. Finally, FAST was demonstrated to achieve FRL-triggered editing of the PLK1 oncogene in a mouse xenograft tumor model. Beyond extending the spectrum of light energies in optogenetic toolbox for CRISPR-Cas9 technologies, this study demonstrates how FAST system can be deployed for programmable deep tissue gene editing in both biological and biomedical contexts toward high precision and spatial specificity.
-
Genome editing for the reproduction and remedy of human diseases in mice.
With the recent progress in genome-editing technologies, such as the CRISPR/Cas9 system, genetically modified animals carrying nucleotide substitutions or large chromosomal rearrangements can be produced rapidly and at low cost. Such genome-editing techniques have been applied in the generation of animal models, especially mice, for reproducing human disease mutations, such as single-nucleotide polymorphisms (SNPs) or large chromosomal rearrangements identified by genome-wide screening analyses. While application methods are under development for various complex mutations involving genome editing for mimicking human disease-causing mutations in mice, functional studies of mouse models carrying replicated human mutations are gradually being published. In this review, we discuss the recent progress in application methods of the CRISPR/Cas9 system, focusing on the production of mouse models of diseases.
-
Rapid Generation of Recombinant Poxviruses Using CRISPR/Cas9 Gene Editing.
The low-frequency natural recombination that is detected in poxvirus-infected cells has long been used to genetically modify poxviruses. Such recombinant poxviruses have found many applications as vaccines for preventing infectious diseases and as experimental cancer therapeutics. Unfortunately, these methods are time consuming, can leave behind "scars" or selectable markers, and many months of work may be required to generate plaque-purified recombinants bearing multiple virus gene substitutions, deletions, and/or inserted transgenes. Over the last decade, several reports have described how CRISPR/Cas9 technologies can be used to better facilitate genetic manipulation of vaccinia virus (VACV). These protocols use Cas9/gRNA complexes to introduce double-stranded breaks into specific sites in virus genomic DNA either in vivo or in vitro. Recombination-repair reactions are then employed to repair the breaks using transfected DNAs encoding the required homologies and desired mutation(s). Here we describe a method where we combine CRISPR/Cas9 genome editing in vitro, followed by Leporipoxvirus-catalyzed repair and reactivation of the cut VACV DNA using repair fragments provided in trans. This method optimizes several steps in the preparation of the CRISPR/Cas9-cut VACV DNA and can be used to introduce mutations at multiple sites without requiring selectable markers. It also provides some guidance regarding how the position of the CRISPR/Cas9-cuts can affect co-conversion of flanking markers embedded in the repair fragment. The method allows researchers to quickly generate recombinant VACV bearing multiple genetic alterations and using only a single round of reactivation and plating.
-
Considerations in engineering viral vectors for genome editing in plants.
Plant viruses have been engineered to express proteins and induce gene silencing for decades. Recently, plant viruses have also been used to deliver components into plant cells for genome editing, a technique called virus-induced genome editing (VIGE). Although more than a dozen plant viruses have been engineered into VIGE vectors and VIGE has been successfully accomplished in some plant species, application of VIGE to crops that are difficult to tissue culture and/or have low regeneration efficiency is still tough. This paper discusses factors to consider for an ideal VIGE vector, including insertion capacity for foreign DNA, vertical transmission ability, expression level of the target gene, stability of foreign DNA insertion, and biosafety. We also proposed a step-by-step schedule for excavating the suitable viral vector for VIGE.
-
Eye on genome editing.
CRISPR/Cas9 genome editing techniques have the potential to treat previously untreatable inherited genetic disorders of vision by correcting mutations that cause these afflictions. Using a prime editor, Qin et al. (2023. J. Exp. Med.https://doi.org/10.1084/jem.20220776) restored visual functions in a mouse model (rd10) of retinitis pigmentosa.
-
A new patient-derived iPSC model for dystroglycanopathies validates a compound that increases glycosylation of α-dystroglycan.
Dystroglycan, an extracellular matrix receptor, has essential functions in various tissues. Loss of α-dystroglycan-laminin interaction due to defective glycosylation of α-dystroglycan underlies a group of congenital muscular dystrophies often associated with brain malformations, referred to as dystroglycanopathies. The lack of isogenic human dystroglycanopathy cell models has limited our ability to test potential drugs in a human- and neural-specific context. Here, we generated induced pluripotent stem cells (iPSCs) from a severe dystroglycanopathy patient with homozygous FKRP (fukutin-related protein gene) mutation. We showed that CRISPR/Cas9-mediated gene correction of FKRP restored glycosylation of α-dystroglycan in iPSC-derived cortical neurons, whereas targeted gene mutation of FKRP in wild-type cells disrupted this glycosylation. In parallel, we screened 31,954 small molecule compounds using a mouse myoblast line for increased glycosylation of α-dystroglycan. Using human FKRP-iPSC-derived neural cells for hit validation, we demonstrated that compound 4-(4-bromophenyl)-6-ethylsulfanyl-2-oxo-3,4-dihydro-1H-pyridine-5-carbonitrile (4BPPNit) significantly augmented glycosylation of α-dystroglycan, in part through upregulation of LARGE1 glycosyltransferase gene expression. Together, isogenic human iPSC-derived cells represent a valuable platform for facilitating dystroglycanopathy drug discovery and therapeutic development.
-
Rapid and efficient generation of GFP-knocked-in Drosophila by the CRISPR-Cas9-mediated genome editing.
The CRISPR-Cas9 technology has been a powerful means to manipulate the genome in a wide range of organisms. A series of GFP knocked-in (GFPKI ) Drosophila strains have been generated through CRISPR-Cas9-induced double strand breaks coupled with homology-directed repairs in the presence of donor plasmids. They visualized specific cell types or intracellular structures in both fixed and live specimen. We provide a rapid and efficient strategy to identify KI lines. This method requires neither co-integration of a selection marker nor prior establishment of sgRNA-expressing transgenic lines. The injection of the mixture of a sgRNA/Cas9 expression plasmid and a donor plasmid into cleavage stage embryos efficiently generated multiple independent KI lines. A PCR-based selection allows to identify KI fly lines at the F1 generation (approximately 4 weeks after injection). These GFPKI strains have been deposited in the Kyoto Drosophila stock center, and made freely available to researchers at non-profit organizations. Thus, they will be useful resources for Drosophila research.
PubMed отдаёт не больше 10 000 первых результатов — уточните запрос, чтобы увидеть остальные.