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 924 из 1000-
Genome Editing with AAV-BR1-CRISPR in Postnatal Mouse Brain Endothelial Cells.
Brain endothelial cells (ECs) are an important component of the blood-brain barrier (BBB) and play key roles in restricting entrance of possible toxic components and pathogens into the brain. However, identifying endothelial genes that regulate BBB homeostasis remains a time-consuming process. Although somatic genome editing has emerged as a powerful tool for discovery of essential genes regulating tissue homeostasis, its application in brain ECs is yet to be demonstrated in vivo. Here, we used an adeno-associated virus targeting brain endothelium (AAV-BR1) combined with the CRISPR/Cas9 system (AAV-BR1-CRISPR) to specifically knock out genes of interest in brain ECs of adult mice. We first generated a mouse model expressing Cas9 in ECs (Tie2 Cas9). We selected endothelial β-catenin (Ctnnb1) gene, which is essential for maintaining adult BBB integrity, as the target gene. After intravenous injection of AAV-BR1-sgCtnnb1-tdTomato in 4-week-old Tie2 Cas9 transgenic mice resulted in mutation of 36.1% of the Ctnnb1 alleles, thereby leading to a dramatic decrease in the level of CTNNB1 in brain ECs. Consequently, Ctnnb1 gene editing in brain ECs resulted in BBB breakdown. Taken together, these results demonstrate that the AAV-BR1-CRISPR system is a useful tool for rapid identification of endothelial genes that regulate BBB integrity in vivo.
-
Optimization of gene editing in cowpea through protoplast transformation and agroinfiltration by targeting the phytoene desaturase gene.
Cowpea (Vigna unguiculata) is a legume staple widely grown across Sub-Saharan Africa and other tropical and sub-tropical regions. Considering projected climate change and global population increases, cowpea's adaptation to hot climates, resistance to drought, and nitrogen-fixing capabilities make it an especially attractive crop for facing future challenges. Despite these beneficial traits, efficient varietal improvement is challenging in cowpea due to its recalcitrance to transformation and long regeneration times. Transient gene expression assays can provide solutions to alleviate these issues as they allow researchers to test gene editing constructs before investing in the time and resource- intensive process of transformation. In this study, we developed an improved cowpea protoplast isolation protocol, a transient protoplast assay, and an agroinfiltration assay to be used for initial testing and validation of gene editing constructs and for gene expression studies. To test these protocols, we assessed the efficacy of a CRISPR-Cas9 construct containing four multiplexed single-guide RNA (sgRNA) sequences using polyethylene glycol (PEG)-mediated transformation and agroinfiltration with phytoene desaturase (PDS) as the target gene. Sanger sequencing of DNA from transformed protoplasts and agroinfiltrated cowpea leaves revealed several large deletions in the target sequences. The protoplast system and agroinfiltration protocol developed in this study provide versatile tools to test gene editing components before initiating plant transformation, thus improving the chance of using active sgRNAs and attaining the desired edits and target phenotype.
-
Generation of Knock-In Syrian Hamsters via Zygote Microinjection Using CRISPR/Cas9 Genome Editing.
Syrian hamsters (Mesocricetus auratus) have long served as valuable model organisms in diverse research fields such as oncology, immunology, and physiology owing to their unique biological and pathological characteristics. Although embryo manipulation techniques such as embryo collection, pronuclear microinjection, and embryo transfer have been established, gene knock-in (KI) hamsters have not yet been reported. Here, we report the successful generation of gene KI Syrian hamsters by microinjecting CRISPR/Cas9 components and plasmid DNA into pronuclear-stage zygotes. Targeted insertion of a DNA cassette up to 8 kb was achieved at the ROSA26 orthologous locus and other genomic sites. Importantly, we confirmed functional expression of a reporter cassette inserted at the ROSA26 site, providing evidence of transcriptional activity at this locus in Syrian hamsters. Furthermore, we demonstrated that frozen-thawed KI embryos could give rise to live offspring using a simplified freezing and thawing protocol originally developed for mice and rats. These results confirm the feasibility and applicability of advanced genome editing technologies in Syrian hamsters. These technological advancements enable the development of versatile KI models for applications such as gene expression monitoring and conditional mutagenesis, thereby expanding the utility of Syrian hamsters as model organisms, comparable to mice and rats.
-
Biolistic-delivery-based transient CRISPR/Cas9 expression enables in planta genome editing in wheat.
The current application of genome editing to crop plants is limited to cultivars that are amenable to in vitro culture and regeneration. Here, we report an in planta genome-editing which does not require callus culture and regeneration. Shoot apical meristems (SAMs) contain a subepidermal cell layer, L2, from which germ cells later develop during floral organogenesis. The biolistic delivery of gold particles coated with plasmids expressing CRISPR/Cas9 components designed to target TaGASR7 were bombarded into SAM-exposed embryos of imbibed seeds. Bombarded embryos showing transient GFP expression within SAM were selected and grown into adult plants. Mutations in the target gene were assessed in fifth-leaf tissue by cleaved amplified polymorphic sequence analysis. Eleven (5.2%) of the 210 bombarded plants carried mutant alleles, and the mutations of three (1.4%) of these were inherited in the next generation. Genotype analysis of T1 plants identified plants homozygous for the three homeologous genes, which were all derived from one T0 plant. These plants showed no detectable integration of the Cas9 and guide RNA genes, indicating that transient expression of CRISPR/Cas9 introduced the mutations. Together, our current method can be used to achieve in planta genome editing in wheat using CRISPR/Cas9 and suggests possible applications to other recalcitrant plant species and variations.
-
CRISPR-Cas9 based gene editing of the immune checkpoint NKG2A enhances NK cell mediated cytotoxicity against multiple myeloma.
Natural Killer (NK) cells are known for their high intrinsic cytotoxic capacity, and the possibility to be applied as 'off-the-shelf' product makes them highly attractive for cell-based immunotherapies. In patients with multiple myeloma (MM), an elevated number of NK cells has been correlated with higher overall-survival rate. However, NK cell function can be impaired by upregulation of inhibitory receptors, such as the immune checkpoint NKG2A. Here, we developed a CRISPR-Cas9-based gene editing protocol that allowed us to knockout about 80% of the NKG2A-encoding killer cell lectin like receptor C1 (KLRC1) locus in primary NK cells. In-depth phenotypic analysis confirmed significant reduction in NKG2A protein expression. Importantly, the KLRC1-edited NK cells showed significantly increased cytotoxicity against primary MM cells isolated from a small cohort of patients, and maintained the NK cell-specific cytokine production. In conclusion, KLRC1-editing in primary NK cells has the prospect of overcoming immune checkpoint inhibition in clinical applications.
-
Introduction of Genome Editing Reagents and Genotyping of Derived Edited Alleles in Soybean (Glycine max (L.) Merr.).
Cas9-based genome editing is a powerful genetic tool for loci specifically targeted for genome modification. This chapter describes up-to-date protocols using Cas9-based genome editing technology, including vector construction with GoldenBraid assembly, Agrobacterium-mediated soybean transformation, and identification of editing in the genome.
-
Efficient breeding of low glutelin content rice germplasm by simultaneous editing multiple glutelin genes via CRISPR/Cas9.
Chronic kidney disease (CKD) and phenylketonuria (PKU) patients need to eat rice with low glutelin content. Therefore, breeding low glutelin content rice varieties with high yield and delicious taste is one of the major goals of rice breeders due to the high demand for the product. In this study, we designed three sgRNAs targeting nine glutelin genes and generated nine T-DNA-free homozygous editing lines with reduced glutelin content compared with the wild-type due to simultaneous mutation(s) in 5-7 glutelin genes. The glutelin content of two lines is even significantly lower than that of the low glutelin content cultivar, LGC-1. Compared to the wild-type, these low glutelin lines showed similar agronomic traits, including yield components and viscosity properties, and can be used as new varieties or parental materials for further breeding.
-
Gene editing vectors for studying nicotinic acetylcholine receptors in cholinergic transmission.
Nicotinic acetylcholine receptors (nAChRs), prototype members of the cys-loop ligand-gated ion channel family, are key mediators of cholinergic transmission in the central nervous system. Despite their importance, technical gaps exist in our ability to dissect the function of individual subunits in the brain. To overcome these barriers, we designed CRISPR/Cas9 small guide RNA sequences (sgRNAs) for the production of loss-of-function alleles in mouse nAChR genes. These sgRNAs were validated in vitro via deep sequencing. We subsequently targeted candidate nAChR genes in vivo by creating herpes simplex virus (HSV) vectors delivering sgRNAs and Cas9 expression to mouse brain. The production of loss-of-function insertions or deletions (indels) by these 'all-in-one' HSV vectors was confirmed using brain slice patch clamp electrophysiology coupled with pharmacological analysis. Next, we developed a scheme for cell type-specific gene editing in mouse brain. Knockin mice expressing Cas9 in a Cre-dependent manner were validated using viral microinjections and genetic crosses to common Cre-driver mouse lines. We subsequently confirmed functional Cas9 activity by targeting the ubiquitous neuronal protein, NeuN, using adeno-associated virus (AAV) delivery of sgRNAs. Finally, the mouse β2 nAChR gene was successfully targeted in dopamine transporter (DAT)-positive neurons via CRISPR/Cas9. The sgRNA sequences and viral vectors, including our scheme for Cre-dependent gene editing, should be generally useful to the scientific research community. These tools could lead to new discoveries related to the function of nAChRs in neurotransmission and behavioral processes.
-
ORFV can carry TRAP gene expression via intracellular CRISPR/Cas9 gene editing technology.
Orf is an acute and highly contracted human and animal infection caused by orf virus (ORFV), which mainly affects sheep, goats, and other species. Clinically, opportunistic or conditional pathogens such as Staphylococcus aureus (S. aureus) are often detected in cases of orf, which greatly increases the risk of disease progression and clinical death. It has been reported that TRAP gene products of S. aureus can broadly influence bacterial life and pathogenicity in vivo, and introduction of exogenous TRAP genes may help to inhibit the proliferation of bacteria. In order to achieve the combined control of ORFV and S. aureus, a novel approach to design a S. aureus TRAP gene vaccine using a live attenuated ORFV vector is proposed. In this study, CRISPR/Cas9 gene editing technology was used to disable vascular endothelial growth factor E of ORFV (VEGF-v) and introduced TRAP gene into this position. TRAP gene expression was detected in keratinocytes infected with recombinant virus. The construction and experimental verification of recombinant ORFV (ORFV-v/TRAP) will provide a reference for in-depth studies on the prevention and control of mixed infectious disease.
-
Identification of Ziziphus jujuba cv. Dongzao DNA Demethylase ZjROS1 Gene Family and Construction of CRISPR/Cas9-Mediated Gene-Editing Vector.
DNA methylation is one of the earliest and most extensively studied epigenetic regulatory mechanisms. The ROS1 (Repressor of Silencing 1) gene was first discovered in Arabidopsis thaliana, and it is a DNA demethylase that can remove 5-methylcytosine from DNA, thereby affecting DNA methylation levels and gene expression. Objectives: The objective of this study was to investigate the role of ROS1 in the development and maturation of Ziziphus jujuba cv. "Dongzao" fruit. Methods: We cloned the ROS1 gene and conducted bioinformatics and expression characteristics analyses on it. Results: Three ROS1 genes, named ZjROS1-1~3, was identified, and each member protein was localized in the nucleus, cytoskeleton, chloroplast, and vacuole. The promoter contained cis-elements such as light response, plant hormone signal transduction, and stress response cis-elements, and it interacted with many proteins such as CMT, MET, and ZDP. The results of the real-time fluorescence quantitative PCR show that ZjROS1 has specific expression patterns in different tissues of Z. jujuba cv. Dongzao, and the expression of ZjROS1-2 in flowers and fruits is high. At the same time, CRISPR/Cas9 technology was used to construct a gene-editing vector for ZjROS1, which provided a basis for the subsequent genetic transformation. Conclusions: In this study, the biological function of ZjROS1 was clarified and a gene-editing vector was constructed, which provided a theoretical basis for the regulation mechanism of demethylase ZjROS1 in the fruit ripening and development of Z. jujuba cv. Dongzao.
PubMed отдаёт не больше 10 000 первых результатов — уточните запрос, чтобы увидеть остальные.