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: 26 105
Страница 148 из 1000-
Live-Animal Epigenome Editing: Convergence of Novel Techniques.
Epigenome editing refers to the generation of precise chromatin alterations and their effects on gene expression and cell biology. Until recently, much of the efforts in epigenome editing were limited to tissue culture models of disease. However, the convergence of techniques from different fields including mammalian genetics, virology, and CRISPR engineering is advancing epigenome editing into a new era. Researchers are increasingly embracing the use of multicellular model organisms to test the role of specific chromatin alterations in development and disease. The challenge of successful live-animal epigenomic editing will depend on a well-informed foundation of the current methodologies for cell-specific delivery and editing accuracy. Here we review the opportunities for basic research and therapeutic applications.
-
In situ functional dissection of RNA cis-regulatory elements by multiplex CRISPR-Cas9 genome engineering.
RNA regulatory elements (RREs) are an important yet relatively under-explored facet of gene regulation. Deciphering the prevalence and functional impact of this post-transcriptional control layer requires technologies for disrupting RREs without perturbing cellular homeostasis. Here we describe genome-engineering based evaluation of RNA regulatory element activity (GenERA), a clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 platform for in situ high-content functional analysis of RREs. We use GenERA to survey the entire regulatory landscape of a 3'UTR, and apply it in a multiplex fashion to analyse combinatorial interactions between sets of miRNA response elements (MREs), providing strong evidence for cooperative activity. We also employ this technology to probe the functionality of an entire MRE network under cellular homeostasis, and show that high-resolution analysis of the GenERA dataset can be used to extract functional features of MREs. This study provides a genome editing-based multiplex strategy for direct functional interrogation of RNA cis-regulatory elements in a native cellular environment.
-
A roadmap toward genome-wide CRISPR screening throughout the organism.
Genome-wide CRISPR screening in the organism has tremendous potential to answer long-standing questions of mammalian physiology and disease. However, bringing this powerful technology in vivo presents unique challenges, including delivering a genome-wide sgRNA library to the appropriate cell type, achieving sufficient coverage of the library, and selecting for the phenotype of interest. In this review, we highlight recent advances in sgRNA delivery, library design, and phenotypic readout that can help overcome these technical challenges and thereby bring high-throughput genetic dissection to an increasing number of tissues and questions. We are excited about the potential for ongoing innovation in these areas to ultimately enable genome-wide CRISPR screening in any cell type of interest in the organism, allowing for unprecedented investigation into diverse questions of mammalian physiology and disease.
-
Promoter Orientation within an AAV-CRISPR Vector Affects Cas9 Expression and Gene Editing Efficiency.
Adeno-associated virus (AAV) vectors have been widely adopted for delivery of CRISPR-Cas components, especially for therapeutic gene editing. For a single vector system, both the Cas9 and guide RNA (gRNA) are encoded within a single transgene, usually from separate promoters. Careful design of this bi-cistronic construct is required due to the minimal packaging capacity of AAV. We investigated how placement of the U6 promoter expressing the gRNA on the reverse strand to SaCas9 driven by a cytomegalovirus promoter affected gene editing rates compared to placement on the forward strand. We show that orientation in the reverse direction reduces editing rates from an AAV vector due to reduced transcription of both SaCas9 and guide RNA. This effect was observed only following AAV transduction; it was not seen following plasmid transfection. These results have implications for the design of AAV-CRISPR vectors, and suggest that results from optimizing plasmid transgenes may not translate when delivered via AAV.
-
Targeted mutagenesis in mice by electroporation of Cpf1 ribonucleoproteins.
-
Efficient induction of haploid plants in wheat by editing of TaMTL using an optimized Agrobacterium-mediated CRISPR system.
The use of CRISPR/LbCpf1 and CRISPR/xCas9 systems in wheat have not yet been reported. In this study, we compared the efficiencies of three CRISPR editing systems (SpCas9, LbCpf1, and xCas9), and three different promoters (OsU6a, TaU3, and TaU6) that drive single-guide (sg)RNA, which were introduced into wheat via Agrobacterium-mediated transformation. The results indicated that TaU3 was a better choice than OsU6a or TaU6. The editing efficiency was higher using two sgRNAs than one sgRNA, and mutants with a large fragment deletion between the two sgRNAs were produced. The LbCpf1 and xCas9 systems could both be used successfully. Two endogenous genes, TaWaxy and TaMTL, were edited with high efficiency by the optimized SpCas9 system, with the highest efficiency (80.5%) being achieved when using TaU3 and two sgRNAs to target TaWaxy. Rates of seed set in the TaMTL-edited T0 transgenic plants were much lower than that of the wild-type. A haploid induction rate of 18.9% was found in the TaMTL-edited T1 plants using the CRISPR/SpCas9 system. Mutants with reverse insertion of the deleted sequences of TaMTL and TaWaxy between the two sgRNAs were identified in the edited T0 plants. In addition, wheat grains lacking embryos or endosperms were observed in the TaMTL-edited T1 generation.
-
What rheumatologists need to know about CRISPR/Cas9.
CRISPR/Cas9 genome editing technology has taken the research world by storm since its use in eukaryotes was first proposed in 2012. Publications describing advances in technology and new applications have continued at an unrelenting pace since that time. In this Review, we discuss the application of CRISPR/Cas9 for creating gene mutations - the application that initiated the current avalanche of interest - and new developments that have largely answered initial concerns about its specificity and ability to introduce new gene sequences. We discuss the new, diverse and rapidly growing adaptations of the CRISPR/Cas9 technique that enable activation, repression, multiplexing and gene screening. These developments have enabled researchers to create sophisticated tools for dissecting the function and inter-relatedness of genes, as well as noncoding regions of the genome, and to identify gene networks and noncoding regions that promote disease or confer disease susceptibility. These approaches are beginning to be used to interrogate complex and multilayered biological systems and to produce complex animal models of disease. CRISPR/Cas9 technology has enabled the application of new therapeutic approaches to treating disease in animal models, some of which are beginning to be seen in the first human clinical trials. We discuss the direct application of these techniques to rheumatic diseases, which are currently limited but are sure to increase rapidly in the near future.
-
CRISPR/Cas9-based genome editing of banana for disease resistance.
Banana production is severely constrained by many pathogens and pests, particularly where a number of them are co-existing. The use of disease-resistant banana varieties is one of the most effective ways to mitigate the negative impacts of pathogens on banana production. Recent advances in new breeding techniques have the potential to accelerate breeding of banana for disease resistance. The CRISPR/Cas9 based genome editing has emerged as the most powerful tool for crop improvement due to its capability of creating precise alterations in plant genome and trait stacking through multiplexing. Recently, the robust CRISPR/Cas9-based genome editing of banana has been established, which can be applied for developing disease-resistant varieties. This article presents a synopsis of recent advancements and perspectives on the application of genome editing for generating disease-resistant banana varieties. It also summarizes the current status of regulatory requirements for the release of genome-edited crop varieties among different countries.
-
Patient-Derived Organoid-Based CRISPR Screens in Cancer Research: Applications, Advances, and Challenges.
Patient-derived organoids (PDOs) have emerged as physiologically relevant cancer models that preserve key genetic, histological, and functional features of the tumors from which they are derived. In parallel, CRISPR-based perturbation technologies have transformed functional genomics by enabling scalable interrogation of gene function. Their integration provides a powerful framework for identifying cancer dependencies, modeling oncogenic evolution, and investigating mechanisms of drug response and resistance in patient-relevant settings. This review examines how CRISPR knockout, CRISPR interference/activation, and precision editing approaches have been applied in PDO systems to uncover context-specific vulnerabilities, reconstruct mutational trajectories, and study tumor heterogeneity. We further compare pooled and arrayed screening formats and discuss what is uniquely enabled by performing CRISPR screens in organoids rather than conventional 2D models. Particular emphasis is placed on the technical and analytical constraints of organoid-based screening, including variable editing efficiency, clonal bottlenecks, biological heterogeneity, and limited scalability. We argue that the major value of organoid-based CRISPR screening lies in its ability to identify functionally actionable cancer vulnerabilities in a patient-contextualized model, while also introducing methodological challenges that must be addressed for robust clinical translation.
-
Generation of ΔF508-CFTR T84 cell lines by CRISPR/Cas9-mediated genome editing.
OBJECTIVES: To provide a simple method to make a stable ΔF508-CFTR-expressing T84 cell line that can be used as an efficient screening model system for ΔF508-CFTR rescue. RESULTS: CFTR knockout cell lines were generated by Cas9 with a single-guide RNA (sgRNA) targeting exon 1 of the CFTR genome, which produced indels that abolished CFTR protein expressions. Next, stable ΔF508-CFTR expression was achieved by genome integration of ΔF508-CFTR via the lentivirus infection system. Finally, we showed functional rescue of ΔF508-CFTR not only by growing the cells at a low temperature, but also incubating with VX-809, a ΔF508-CFTR corrector, in the established T84 cells expressing ΔF508-CFTR. CONCLUSIONS: This cell system provides an appropriate screening platform for rescue of ΔF508-CFTR, especially related to protein folding, escaped from endoplasmic-reticulum-associated protein degradation, and membrane transport.
PubMed отдаёт не больше 10 000 первых результатов — уточните запрос, чтобы увидеть остальные.