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: 26 220
Страница 750 из 1000-
Electroporation of sheep zygotes as an alternative to microinjection for the generation of CRISPR/Cas genome edited models.
Zygote microinjection is considered the most suitable technique to introduce CRISPR/Cas9 reagents for efficient genome editing in livestock. In this study, zygote electroporation was evaluated as an alternative to microinjection for CRISPR/Cas9-mediated genome editing in sheep. Four experiments were conducted on 3548 cumulus-oocyte complexes. Acid Tyrode's solution (AT) was used to partially degrade the zona pellucida (ZP) to improve reagent entry, resulting in ZP thinning with longer AT exposure (P < 0.05). Although early embryo development was impaired by AT exposure (P < 0.05), blastocyst rates were similar across all groups by day 8. Electroporation conditions were optimized by testing pulse length (1 or 3 ms), with the best results from 6 pulses of 20 V for 3 ms with AT during 60 s. Electroporation with 500 ng/μL Cas9 and 300 ng/μL sgRNA with AT during 60 s achieved a 38.5 % mutation rate. When compared with conventional microinjection, electroporation had higher developmental rates but a lower mutation rate (21.4 % vs. 60.0 %; P < 0.05). These findings suggest that electroporation is a viable, cost-effective technique for genome editing in sheep. Nevertheless, further research will be required to fine-tune electroporation conditions and enhance efficiency in terms of mutation rate.
-
Protocol of Phytophthora capsici Transformation Using the CRISPR-Cas9 System.
Phytophthora capsici is an important plant pathogen, which causes significant economic losses on multiple vegetable crops worldwide. It is an ideal model pathogen to study the role of important genes, plant-pathogen interactions, and fungicide resistance mechanisms etc. due to its wide range of hosts and genetic diversity. A more efficient gene editing tool is required to do these studies. Here, we describe a detailed experimental procedure using the CRISPR-Cas9 system to edit genes of interest in P. capsici, which has been proven to be an accurate and efficient gene editing method in P. capsici.
-
Senescence and inflammation are unintended adverse consequences of CRISPR-Cas9/AAV6-mediated gene editing in hematopoietic stem cells.
Gene editing (GE) using homology-directed repair (HDR) in hematopoietic stem and progenitor cells (HSPCs) offers promise for long-range gene correction of inherited genetic disorders. However, cellular responses induced by CRISPR-Cas9/AAV6 engineering impair the long-term repopulating potential of HDR-edited HSPCs, adversely impacting the safety and efficacy of clinical translation. Our study uncovers a durable senescence-like response in genetically engineered HSPCs triggered by p53 and interleukin (IL)-1/nuclear factor κB (NF-κB) activation, which restricts graft size and clonal diversity in long-term transplantation assays. We show that transient p53 inhibition or blocking inflammatory pathways mitigates senescence-associated responses, improving the repopulating capacity of edited HSPCs. Importantly, we identify treatment with Anakinra, an IL-1 signaling antagonist, as a promising strategy to enhance polyclonal output in HDR-edited cells while minimizing genotoxicity risks associated with the editing procedure. Overall, our findings present strategies to overcome key hurdles in HDR-based HSPC gene therapies, providing a framework for enhancing their efficacy and safety in clinical applications.
-
CRISPR/Cas-Mediated Genome Editing of Streptomyces.
Streptomyces are an important source and reservoir of natural products with diverse applications in medicine, agriculture, and food. Engineered Streptomyces strains have also proven to be functional chassis for the discovery and production of bioactive compounds and enzymes. However, genetic engineering of Streptomyces is often laborious and time-consuming. Here we describe protocols for CRISPR/Cas-mediated genome editing of Streptomyces. Starting from the design and assembly of all-in-one CRISPR/Cas constructs for efficient double-strand break-mediated genome editing, we also present protocols for intergeneric conjugation, CRISPR/Cas plasmid curing, and validation of edited strains.
-
Refining strategies to translate genome editing to the clinic.
Recent progress in developing programmable nucleases, such as zinc-finger nucleases, transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeat (CRISPR)-Cas nucleases, have paved the way for gene editing to enter clinical practice. This translation is a result of combining high nuclease activity with high specificity and successfully applying this technology in various preclinical disease models, including infectious disease, primary immunodeficiencies, hemoglobinopathies, hemophilia and muscular dystrophy. Several clinical gene-editing trials, both ex vivo and in vivo, have been initiated in the past 2 years, including studies that aim to knockout genes as well as to add therapeutic transgenes. Here we discuss the advances made in the gene-editing field in recent years, and specify priorities that need to be addressed to expand therapeutic genome editing to further disease entities.
-
The Development of Herbicide Resistance Crop Plants Using CRISPR/Cas9-Mediated Gene Editing.
The rapid increase in herbicide-resistant weeds creates a huge challenge to global food security because it can reduce crop production, causing considerable losses. Combined with a lack of novel herbicides, cultivating herbicide-resistant crops becomes an effective strategy to control weeds because of reduced crop phytotoxicity, and it expands the herbicidal spectrum. Recently developed clustered regularly interspaced short palindromic repeat/CRISPR-associated protein (CRISPR/Cas)-mediated genome editing techniques enable efficiently targeted modification and hold great potential in creating desired plants with herbicide resistance. In the present review, we briefly summarize the mechanism responsible for herbicide resistance in plants and then discuss the applications of traditional mutagenesis and transgenic breeding in cultivating herbicide-resistant crops. We mainly emphasize the development and use of CRISPR/Cas technology in herbicide-resistant crop improvement. Finally, we discuss the future applications of the CRISPR/Cas system for developing herbicide-resistant crops.
-
Focused ultrasound and microbubble-mediated delivery of CRISPR-Cas9 ribonucleoprotein to human induced pluripotent stem cells.
CRISPR-Cas9 ribonucleoproteins (RNPs) have been heavily considered for gene therapy due to their high on-target efficiency, rapid activity, and lack of insertional mutagenesis relative to other CRISPR-Cas9 delivery formats. Genetic diseases such as hypertrophic cardiomyopathy currently lack effective treatment strategies and are prime targets for CRISPR-Cas9 gene editing technology. However, current in vivo delivery strategies for Cas9 pose risks of unwanted immunogenic responses. This proof-of-concept study aimed to demonstrate that focused ultrasound (FUS) in combination with microbubbles can be used to deliver Cas9-sgRNA (single-guide RNA) RNPs and functionally edit human induced pluripotent stem cells (hiPSCs) in vitro, a model system that can be expanded to cardiovascular research via hiPSC-derived cardiomyocytes. Here, we first determine acoustic conditions suitable for the viable delivery of large proteins to hiPSCs with clinical Definity microbubble agents using our customized experimental platform. From here, we delivered Cas9-sgRNA RNP complexes targeting the EGFP (enhanced green fluorescent protein) gene to EGFP-expressing hiPSCs for EGFP knockout. Simultaneous acoustic cavitation detection during treatment confirmed a strong correlation between microbubble disruption and viable FUS-mediated protein delivery in hiPSCs. This study shows for the first time the potential for an FUS-mediated technique for targeted and precise CRISPR-Cas9 gene editing in human stem cells.
-
Research Progress on Nanoparticles-Based CRISPR/Cas9 System for Targeted Therapy of Tumors.
Cancer is a genetic mutation disease that seriously endangers the health and life of all human beings. As one of the most amazing academic achievements in the past decade, CRISPR/Cas9 technology has been sought after by many researchers due to its powerful gene editing capability. CRISPR/Cas9 technology shows great potential in oncology, and has become one of the most promising technologies for cancer genome-editing therapeutics. However, its efficiency and the safety issues of in vivo gene editing severely limit its widespread application. Therefore, developing a suitable delivery method for the CRISPR/Cas9 system is an urgent problem to be solved at present. Rapid advances in nanomedicine suggest nanoparticles could be a viable option. In this review, we summarize the latest research on the potential use of nanoparticle-based CRISPR/Cas9 systems in cancer therapeutics, in order to further their clinical application. We hope that this review will provide a novel insight into the CRISPR/Cas9 system and offer guidance for nanocarrier designs that will enable its use in cancer clinical applications.
-
Optimization of Vectors and Targeting Strategies Including GoldenBraid and Genome Editing Tools: GoldenBraid Assembly of Multiplex CRISPR /Cas12a Guide RNAs for Gene Editing in Nicotiana benthamiana.
New breeding techniques, especially CRISPR/Cas, could facilitate the expansion and diversification of molecular farming crops by speeding up the introduction of new traits that improve their value as biofactories. One of the main advantages of CRISPR/Cas is its ability to target multiple loci simultaneously, a key feature known as multiplexing. This characteristic is especially relevant for polyploid species, as it is the case of Nicotiana benthamiana and other species of the same genus widely used in molecular farming. Here, we describe in detail the making of a multiplex DNA construct for genome editing in N. benthamiana using the GoldenBraid modular cloning platform. In this case, the procedure is adapted for the requirements of LbCas12a (Lachnospiraceae bacterium Cas12a), a nuclease whose cloning strategy differs from that of the more often used SpCas9 (Streptococcus pyogenes Cas9) enzyme. LbCas12a-mediated edition has several advantages, as its high editing efficiency, described for different plant species, and its T/A-rich PAM sequence, which expands the range of genomic loci that can be targeted by site-specific nucleases. The protocol also includes recommendations for the selection of protospacer sequences and indications for the analysis of editing results.
-
Comparison of dystrophin expression following gene editing and gene replacement in an aged preclinical DMD animal model.
Gene editing has shown promise for correcting or bypassing dystrophin mutations in Duchenne muscular dystrophy (DMD). However, preclinical studies have focused on young animals with limited muscle fibrosis and wasting, thereby favoring muscle transduction, myonuclear editing, and prevention of disease progression. Here, we explore muscle-specific dystrophin gene editing following intramuscular delivery of AAV6:CK8e-CRISPR/SaCas9 in 3- and 8-year-old dystrophic CXMD dogs and provide a qualitative comparison to AAV6:CK8e-micro-dystrophin gene replacement at 6 weeks post-treatment. Gene editing restored the dystrophin reading frame in ∼1.3% of genomes and in up to 4.0% of dystrophin transcripts following excision of a 105-kb mutation containing region spanning exons 6-8. However, resulting dystrophin expression levels and effects on muscle pathology were greater with the use of micro-dystrophin gene transfer. This study demonstrates that our muscle-specific multi-exon deletion strategy can correct a frequently mutated region of the dystrophin gene in an aged large animal DMD model, but underscores that further enhancements are required to reach efficiencies comparable to AAV micro-dystrophin. Our observations also indicate that treatment efficacy and state of muscle pathology at the time of intervention are linked, suggesting the need for additional methodological optimizations related to age and disease progression to achieve relevant clinical translation of CRISPR-based therapies to all DMD patients.
PubMed отдаёт не больше 10 000 первых результатов — уточните запрос, чтобы увидеть остальные.