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 901 из 1000-
Seeing is believing: a visualization toolbox to enhance selection efficiency in maize genome editing.
-
Inducible RNA targeting and N6-methyladenosine editing by a split-Cas13 architecture.
-
Direct detection of CRISPR-Cas9 ribonucleoprotein gene doping using RNA immunoprecipitation and quantitative PCR.
Gene doping, using technologies such as CRISPR-Cas9, poses a considerable threat to the integrity of sports. In 2018, the World Anti-Doping Agency implemented a ban on genome editing, which highlighted the need for sensitive and specific detection methods. Detection techniques that are currently available have shown effectiveness in specific contexts, but are limited by low sensitivity and short detection windows. To overcome these limitations, this study presents a new detection method for CRISPR-Cas9 ribonucleoprotein (RNP) complexes, termed RNA immunoprecipitation followed by quantitative PCR (RIP-qPCR). The primary focus of this research was the in vitro development of a detection method targeting genes critical for doping, including myostatin (MSTN), α-actinin 3 (ACTN3), erythropoietin receptor (EPOR), and erythropoietin (EPO), with in vivo proof-of-concept demonstrated using MSTN. The RIP-qPCR method demonstrated sensitive performance, with a limit of quantification of 0.1 ng/mL in plasma. This method successfully detected single guide RNA targeting MSTN, ACTN3, EPOR, and EPO, along with two types of Cas9 proteins in RNP complexes in vitro. Additionally, the detection capabilities of RIP-qPCR were maintained for up to 30 days when plasma samples were stored at 4 °C. In vivo experiments were performed where RNPs were administered via intramuscular and intravenous injections to target the murine Mstn gene. CRISPR-Cas9 RNPs remained detectable for up to 24 h following intramuscular injection and 12 h after intravenous injection. This study underscores the potential of RIP-qPCR as a powerful tool for anti-doping analysis, with future efforts on expanding the target gene panel to enhance the detection of gene editing in sports doping.
-
CRISPR/Cas9-mediated multiplex gene editing of gamma and omega gliadins: paving the way for gliadin-free wheat.
Wheat is a staple cereal in the human diet. Despite its significance, an increasing percentage of the population suffers adverse reactions to wheat, which are triggered by wheat gluten, particularly the gliadin fractions. In this study, we employed CRISPR/Cas [clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein] multiplexing to introduce targeted mutations into γ- and ω-gliadin genes of wheat, to produce lines deficient in one or both immunogenic gliadin fractions simultaneously. For this study, eight single guide RNAs (sgRNAs) were designed and combined into four plasmids to produce 59 modified wheat lines, of which 20 exhibited mutations in the target genes. Characterization of these lines through Sanger sequencing or next-generation sequencing revealed a complex pattern of InDels, including deletions spanning multiple sgRNAs. The mutations were transmitted to the offspring, and the analysis of homozygous derived lines by reverse-phase HPLC and monoclonal antibodies showed a 97.7% reduction in gluten content. Crossing these lines with other CRISPR/Cas lines deficient in the α-gliadins allowed multiple mutations to be combined. This work represents an important step forward in the use of CRISPR/Cas to develop gluten-free wheat.
-
Protoplast technology enables the identification of efficient multiplex genome editing tools in Phalaenopsis.
Phalaenopsis orchids are popular ornamental plants worldwide. The application and optimization of efficient CRISPR-Cas genome editing toolkits in Phalaenopsis greatly accelerate the development of orchid gene function and breeding research. However, these methods are greatly hindered by the deficiency of a rapid screening system. In this study, we established a fast and convenient Phalaenopsis protoplast technology for the identification of functional genome editing tools. Two multiplex genome editing tools, PTG-Cas9-HPG (PTG, polycistronic tRNA-gRNA) system and RMC-Cpf1-HPG (RMC, ribozyme-based multi-crRNA) system, were developed for Phalaenopsis genome editing and further evaluated by established protoplast technology. We successfully detected various editing events comprising substitution and indel at designed target sites of the PDS gene and MADS gene, showing that both PTG-Cas9-HPG and RMC-Cpf1-HPG multiplex genome editing systems are functional in Phalaenopsis. Additionally, by optimizing the promoter that drives Cpf1 expression, we found that Super promoter can significantly improve the editing efficiency of the RMC-Cpf1-HPG system. Altogether, we successfully developed two efficient multiplex genome editing systems, PTG-Cas9-HPG and RMC-Cpf1-HPG, for Phalaenopsis, and the established protoplast-based screening technology provides a valuable foundation for developing more diverse and efficient genome editing toolkits and facilitating the development of orchid precision breeding.
-
Efficient CRISPR/Cas12a-Based Genome-Editing Toolbox for Metabolic Engineering in Methanococcus maripaludis.
The rapid-growing and genetically tractable methanogen Methanococcus maripaludis is a promising host organism for the biotechnological conversion of carbon dioxide and renewable hydrogen to fuels and value-added products. Expansion of its product scope through metabolic engineering necessitates reliable and efficient genetic tools, particularly for genome edits that affect the primary metabolism and cell growth. Here, we have designed a genome-editing toolbox by utilizing Cas12a from Lachnospiraceae bacterium ND2006 (LbCas12a) in combination with the homology-directed repair machinery endogenously present in M. maripaludis. This toolbox can delete target genes with a success rate of up to 95%, despite the hyperpolyploidy of M. maripaludis. For the purpose of demonstrating a large deletion, the M. maripaludis flagellum operon (∼8.9 kbp) was replaced by the Escherichia coli β-glucuronidase gene. To facilitate metabolic engineering and flux balancing in M. maripaludis, the relative strength of 15 different promoters was quantified in the presence of two common growth substrates, either formate or carbon dioxide and hydrogen. This CRISPR/LbCas12a toolbox can be regarded as a reliable and quick method for genome editing in a methanogen.
-
A Dual sgRNA Approach for Functional Genomics in Arabidopsis thaliana.
Reverse genetics uses loss-of-function alleles to interrogate gene function. The advent of CRISPR/Cas9-based gene editing now allows the generation of knock-out alleles for any gene and entire gene families. Even in the model plant Arabidopsis thaliana, gene editing is welcomed as T-DNA insertion lines do not always generate null alleles. Here, we show efficient generation of heritable mutations in Arabidopsis using CRISPR/Cas9 with a workload similar to generating overexpression lines. We obtain for several different genes Cas9 null-segregants with bi-allelic mutations in the T2 generation. While somatic mutations were predominantly generated by the canonical non-homologous end joining (cNHEJ) pathway, we observed inherited mutations that were the result of synthesis-dependent microhomology-mediated end joining (SD-MMEJ), a repair pathway linked to polymerase θ (PolQ). We also demonstrate that our workflow is compatible with a dual sgRNA approach in which a gene is targeted by two sgRNAs simultaneously. This paired nuclease method results in more reliable loss-of-function alleles that lack a large essential part of the gene. The ease of the CRISPR/Cas9 workflow should help in the eventual generation of true null alleles of every gene in the Arabidopsis genome, which will advance both basic and applied plant research.
-
Transcriptional regulation with CRISPR-Cas9: principles, advances, and applications.
CRISPR-Cas9 has recently emerged as a promising system for multiplexed genome editing as well as epigenome and transcriptome perturbation. Due to its specificity, ease of use and highly modular programmable nature, it has been widely adopted for a variety of applications such as genome editing, transcriptional inhibition and activation, genetic screening, DNA localization imaging, and many more. In this review, we will discuss non-editing applications of CRISPR-Cas9 for transcriptome perturbation, metabolic engineering, and synthetic biology.
-
Efficient Genome Editing of a Facultative Thermophile Using Mesophilic spCas9.
Well-developed genetic tools for thermophilic microorganisms are scarce, despite their industrial and scientific relevance. Whereas highly efficient CRISPR/Cas9-based genome editing is on the rise in prokaryotes, it has never been employed in a thermophile. Here, we apply Streptococcus pyogenes Cas9 (spCas9)-based genome editing to a moderate thermophile, i.e., Bacillus smithii, including a gene deletion, gene knockout via insertion of premature stop codons, and gene insertion. We show that spCas9 is inactive in vivo above 42 °C, and we employ the wide temperature growth range of B. smithii as an induction system for spCas9 expression. Homologous recombination with plasmid-borne editing templates is performed at 45-55 °C, when spCas9 is inactive. Subsequent transfer to 37 °C allows for counterselection through production of active spCas9, which introduces lethal double-stranded DNA breaks to the nonedited cells. The developed method takes 4 days with 90, 100, and 20% efficiencies for gene deletion, knockout, and insertion, respectively. The major advantage of our system is the limited requirement for genetic parts: only one plasmid, one selectable marker, and a promoter are needed, and the promoter does not need to be inducible or well-characterized. Hence, it can be easily applied for genome editing purposes in both mesophilic and thermophilic nonmodel organisms with a limited genetic toolbox and ability to grow at, or tolerate, temperatures of 37 and at or above 42 °C.
-
Versatile electroporation protocols enable reproducible CRISPR-RNP delivery across multiple primary mouse cells of the hematopoietic lineage.
Genetic engineering of primary hematopoietic cells is essential for mechanistic immunology studies, however the development of cell-based therapies yet remains constrained by two major factors: the fragility of many primary lineages and challenges of viral delivery platforms that are costly, time-intensive, and biologically confounding. Here, we optimized scalable CRISPR-Cas9 electroporation workflows using the ExPERT platform across three primary mouse hematopoietic cell types: OT-I CD8+ T cells, bone marrow-derived macrophages (BMDMs), and hematopoietic stem/progenitor cells (HSPCs). In activated OT-I CD8+ T cells, two electroporation programs supported high-efficiency mRNA and RNP delivery with minimal impact on cell viability or proliferative capacity, with subtle activation-state-dependent sensitivity at higher energy settings. Extending optimization to myeloid and stem cell lineages, we found that BMDMs maintained high viability following electroporation, and a high-performing electroporation program supported robust RNP delivery and efficient target gene knockout while preserving macrophage differentiation. In HSPCs, the same program enabled consistent RNP delivery, sustained viability, and reproducible gene knockout. Together, these findings establish ExPERT electroporation as a robust, reproducible, and modular platform for genome editing across primary mouse hematopoietic lineages, lowering barriers to rapid genetic perturbation for both discovery and translational applications. This manuscript has been posted on BioRxiv (https://www.biorxiv.org/content/10.64898/2026.01.27.702081v1).
PubMed отдаёт не больше 10 000 первых результатов — уточните запрос, чтобы увидеть остальные.