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 896 из 1000-
Protocol for the functional evaluation of genetic variants using saturation genome editing.
Saturation genome editing (SGE) employs CRISPR-Cas9 and homology-directed repair (HDR) to introduce exhaustive nucleotide modifications at specific genomic sites in multiplex, enabling the functional analysis of genetic variants while preserving their native genomic context. Here, we present a protocol for SGE-based variant evaluation in HAP1-A5 cells. We describe the steps for designing variant libraries, single-guide RNAs (sgRNAs), and oligonucleotide primers for PCR. We also detail the sample preparation before the SGE screen, the cellular screening process, and subsequent next-generation sequencing (NGS) library preparation. For complete details on the use and execution of this protocol, please refer to Radford et al.,1 Waters et al.,2 and Olvera-León et al.3.
-
Genome editing with CRISPR/Cas9 in Pinus radiata (D. Don).
BACKGROUND: To meet increasing demand for forest-based products and protect natural forests from further deforestation requires increased productivity from planted forests. Genetic improvement of conifers by traditional breeding is time consuming due to the long juvenile phase and genome complexity. Genetic modification (GM) offers the opportunity to make transformational changes in shorter time frames but is challenged by current genetically modified organism (GMO) regulations. Genome editing, which can be used to generate site-specific mutations, offers the opportunity to rapidly implement targeted improvements and is globally regulated in a less restrictive way than GM technologies. RESULTS: We have demonstrated CRISPR/Cas9 genome editing in P. radiata targeting a single-copy cell wall gene GUX1 in somatic embryogenic tissue and produced plantlets from the edited tissue. We generated biallelic INDELs with an efficiency of 15 % using a single gRNA. 12 % of the transgenic embryogenic tissue was edited when two gRNAs were used and deletions of up to 1.3 kb were identified. However, the regenerated plants did not contain large deletions but had single nucleotide insertions at one of the target sites. We assessed the use of CRISPR/Cas9 ribonucleoproteins (RNPs) for their ability to accomplish DNA-free genome editing in P. radiata. We chose a hybrid approach, with RNPs co-delivered with a plasmid-based selectable marker. A two-gRNA strategy was used which produced an editing efficiency of 33 %, and generated INDELs, including large deletions. Using the RNP approach, deletions found in embryogenic tissue were also present in the plantlets. But, all plants produced using the RNP strategy were monoallelic. CONCLUSIONS: We have demonstrated the generation of biallelic and monoallelic INDELs in the coniferous tree P. radiata with the CRISPR/Cas9 system using plasmid expressed Cas9 gRNA and RNPs respectively. This opens the opportunity to apply genome editing in conifers to rapidly modify key traits of interest.
-
mTREX2 co-expression promotes large deletions in CRISPR/Cas12a editing of the highly polyploid sugarcane genome.
The LbCas12ai-mTREX2 system enhances large deletions, and the LbCas12ai-RRV enables efficient editing of lw1 in sugarcane. Sugarcane (Saccharum spp. hybrids) is a major feedstock for biofuel production and the primary source of global table sugar; however, its highly polyploid and complex genome constrains the effective deployment of functional genomics and precision breeding strategies. While CRISPR/Cas9-mediated genome editing has been reported in sugarcane, Cas12a-based editing remains unexplored despite its advantages in targeting AT-rich genomic regions. In this study, we report efficient CRISPR/Cas12a-mediated targeted mutagenesis of the lw1 gene in sugarcane. The lemon white1 (lw1) is a key regulator of chlorophyll and carotenoid biosynthesis, and targeted lw1 mutagenesis results in a visible phenotype. We compared the editing performance of intron-containing LbCas12a-R (LbCas12ai-R) and LbCas12a-RRV (LbCas12ai-RRV) variants and assessed the impact of Mus musculus Three prime repair exonuclease 2 (mTREX2) co-expression on editing efficiency and deletion profiles. Editing efficiencies ranged from 31 to 85%, where LbCas12ai-RRV consistently outperformed LbCas12ai-R in both overall editing efficiency and allelic editing efficiency. Co-expression of the mTREX2 shifted the mutation spectrum toward larger deletions, increasing the frequency of moderate-to-large deletions. The ≥ 51 bp deletion class increased ~7-fold in both variants. Capillary electrophoresis-based genotyping provided a cost-effective, high-throughput alternative to next-generation sequencing (NGS) and accurately predicted allelic editing efficiency and indel size. This first report of CRISPR/Cas12a-mediated genome editing in sugarcane provides a framework for future engineering of AT-rich cis-regulatory elements in the polyploid sugarcane genome.
-
Self-cleaving guide RNAs enable pharmacological selection of precise gene editing events in vivo.
Expression of guide RNAs in the CRISPR/Cas9 system typically requires the use of RNA polymerase III promoters, which are not cell-type specific. Flanking the gRNA with self-cleaving ribozyme motifs to create a self-cleaving gRNA overcomes this limitation. Here, we use self-cleaving gRNAs to create drug-selectable gene editing events in specific hepatocyte loci. A recombinant Adeno Associated Virus vector targeting the Albumin locus with a promoterless self-cleaving gRNA to create drug resistance is linked in cis with the therapeutic transgene. Gene expression of both are dependent on homologous recombination into the target locus. In vivo drug selection for the precisely edited hepatocytes allows >30-fold expansion of gene-edited cells and results in therapeutic levels of a human Factor 9 transgene. Importantly, self-cleaving gRNA expression is also achieved after targeting weak hepatocyte genes. We conclude that self-cleaving gRNAs are a powerful system to enable cell-type specific in vivo drug resistance for therapeutic gene editing applications.
-
Switching the activity of Cas12a using guide RNA strand displacement circuits.
The CRISPR effector protein Cas12a has been used for a wide variety of applications such as in vivo gene editing and regulation or in vitro DNA sensing. Here, we add programmability to Cas12a-based DNA processing by combining it with strand displacement-based reaction circuits. We first establish a viable strategy for augmenting Cas12a guide RNAs (gRNAs) at their 5' end and then use such 5' extensions to construct strand displacement gRNAs (SD gRNAs) that can be activated by single-stranded RNA trigger molecules. These SD gRNAs are further engineered to exhibit a digital and orthogonal response to different trigger RNA inputs-including full length mRNAs-and to function as multi-input logic gates. We also demonstrate that SD gRNAs can be designed to work inside bacterial cells. Using such in vivo SD gRNAs and a DNase inactive version of Cas12a (dCas12a), we demonstrate logic gated transcriptional control of gene expression in E. coli.
-
DGAT2 stability is increased in response to DGAT1 inhibition in gene edited HepG2 cells.
In eukaryotic organisms, two unrelated acyl-CoA:diacylglycerol acyltransferase (DGAT) enzymes, DGAT1 and DGAT2, catalyze the final step of the triacylglycerol biosynthetic pathway. Both enzymes are highly expressed in lipogenic tissues, such as adipose tissue, small intestine and the liver. DGAT2 has a prominent role in hepatocyte lipid metabolism synthesizing triacylglycerols that are utilized for very low-density lipoprotein assembly. However, due to the lack of useful antibodies to detect endogenous DGAT2 protein, it has been difficult to determine how this enzyme functions at the cellular level. We have unsuccessfully tested many commercial antibodies as well as our own "in-house" antibodies. There is currently no evidence that DGAT2 undergoes processing such that antigenic epitopes to these antibodies are removed. As an alternative, many studies have utilized epitope tagged versions of DGAT2 overexpressed in cells. These approaches can provide valuable information about a protein, but can be subject to artifacts, such as mislocalization, misregulation, protein aggregation and abnormal protein-protein interactions. In this study, we used gene editing with CRISPR/Cas9 to add three consecutive FLAG epitopes to the C-terminus of endogenous DGAT2 in HepG2 cells. HepG2 cells, derived from a human hepatocellular carcinoma, have been routinely used as a cell model to study human hepatocyte lipid and lipoprotein metabolism. Using this system allowed us to successfully detect DGAT2 expressed from its endogenous locus in HepG2 cells by immunoblotting with anti-FLAG antibodies.
-
When Less Is More: Targeting the Myostatin Gene in Livestock for Augmenting Meat Production.
How to increase meat production is one of the main questions in animal breeding. Selection for improved body weight has been made and, due to recent genomic advances, naturally occurring variants that are responsible for controlling economically relevant phenotypes have been revealed. The myostatin (MSTN) gene, a superstar gene in animal breeding, was discovered as a negative controller of muscle mass. In some livestock species, natural mutations in the MSTN gene could generate the agriculturally desirable double-muscling phenotype. However, some other livestock species or breeds lack these desirable variants. Genetic modification, particularly gene editing, offers an unprecedented opportunity to induce or mimic naturally occurring mutations in livestock genomes. To date, various MSTN-edited livestock species have been generated using different gene modification tools. These MSTN gene-edited models have higher growth rates and increased muscle mass, suggesting the high potential of utilizing MSTN gene editing in animal breeding. Additionally, post-editing investigations in most livestock species support the favorable influence of targeting the MSTN gene on meat quantity and quality. In this Review, we provide a collective discussion on targeting the MSTN gene in livestock to further encourage its utilization opportunities. It is expected that, shortly, MSTN gene-edited livestock will be commercialized, and MSTN-edited meat will be on the tables of ordinary customers.
-
Integration, abundance, and transmission of mutations and transgenes in a series of CRISPR/Cas9 soybean lines.
BACKGROUND: As with many plant species, current genome editing strategies in soybean are initiated by stably transforming a gene that encodes an engineered nuclease into the genome. Expression of the transgene results in a double-stranded break and repair at the targeted locus, oftentimes resulting in mutation(s) at the intended site. As soybean is a self-pollinating species with 20 chromosome pairs, the transgene(s) in the T0 plant are generally expected to be unlinked to the targeted mutation(s), and the transgene(s)/mutation(s) should independently assort into the T1 generation, resulting in Mendellian combinations of transgene presence/absence and allelic states within the segregating family. This prediction, however, is not always consistent with observed results. RESULTS: In this study, we investigated inheritance patterns among three different CRISPR/Cas9 transgenes and their respective induced mutations in segregating soybean families. Next-generation resequencing of four T0 plants and four T1 progeny plants, followed by broader assessments of the segregating families, revealed both expected and unexpected patterns of inheritance among the different lineages. These unexpected patterns included: (1) A family in which T0 transgenes and mutations were not transmitted to progeny; (2) A family with four unlinked transgene insertions, including two respectively located at paralogous CRISPR target break sites; (3) A family in which mutations were observed and transmitted, but without evidence of transgene integration nor transmission. CONCLUSIONS: Genome resequencing provides high-resolution of transgene integration structures and gene editing events. Segregation patterns of these events can be complicated by several potential mechanisms. This includes, but is not limited to, plant chimeras, multiple unlinked transgene integrations, editing of intended and paralogous targets, linkage between the transgene integration and target site, and transient expression of the editing reagents without transgene integration into the host genome.
-
CRISPR-Cas: a game-changer in vaccine development and the fight against viral infections.
Understanding the mechanisms of virus-host interactions is crucial for advancing our knowledge of viral pathogenesis and developing effective antiviral strategies. As novel, highly virulent pathogens continue to emerge, overcoming the challenges in virus-host biology and infection control is more important than ever. The CRISPR-Cas system has revolutionized genetic engineering, offering a precise and efficient application in synthetic biology. It is also invaluable in virology research, diagnostics, and antiviral therapeutics. In this review, we explore the latest advances in CRISPR-Cas-based approaches to elucidate virus-host interactions, focusing on the identification of key receptors and host factors essential for virus infection. Furthermore, we discuss the potential of the CRISPR-Cas system as an antiviral strategy for tackling newly emerging and re-emerging viruses. Finally, we discuss the potential of CRISPR-Cas-based gene editing for the development of innovative viral vaccines.
-
Clinical applications of the CRISPR/Cas9 genome-editing system: Delivery options and challenges in precision medicine.
CRISPR/Cas9 is an effective gene editing tool with broad applications for the prevention or treatment of numerous diseases. It depends on CRISPR (clustered regularly interspaced short palindromic repeats) as a bacterial immune system and plays as a gene editing tool. Due to the higher specificity and efficiency of CRISPR/Cas9 compared to other editing approaches, it has been broadly investigated to treat numerous hereditary and acquired illnesses, including cancers, hemolytic diseases, immunodeficiency disorders, cardiovascular diseases, visual maladies, neurodegenerative conditions, and a few X-linked disorders. CRISPR/Cas9 system has been used to treat cancers through a variety of approaches, with stable gene editing techniques. Here, the applications and clinical trials of CRISPR/Cas9 in various illnesses are described. Due to its high precision and efficiency, CRISPR/Cas9 strategies may treat gene-related illnesses by deleting, inserting, modifying, or blocking the expression of specific genes. The most challenging barrier to the in vivo use of CRISPR/Cas9 like off-target effects will be discussed. The use of transfection vehicles for CRISPR/Cas9, including viral vectors (such as an Adeno-associated virus (AAV)), and the development of non-viral vectors is also considered.
PubMed отдаёт не больше 10 000 первых результатов — уточните запрос, чтобы увидеть остальные.