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: 26 233
Страница 846 из 1000-
Addressing the dark matter of gene therapy: technical and ethical barriers to clinical application.
Gene therapies for genetic diseases have been sought for decades, and the relatively recent development of the CRISPR/Cas9 gene-editing system has encouraged a new wave of interest in the field. There have nonetheless been significant setbacks to gene therapy, including unintended biological consequences, ethical scandals, and death. The major focus of research has been on technological problems such as delivery, potential immune responses, and both on and off-target effects in an effort to avoid negative clinical outcomes. While the field has concentrated on how we can better achieve gene therapies and gene editing techniques, there has been less focus on when and why we should use such technology. Here we combine discussion of both the technical and ethical barriers to the widespread clinical application of gene therapy and gene editing, providing a resource for gene therapy experts and novices alike. We discuss ethical problems and solutions, using cystic fibrosis and beta-thalassemia as case studies where gene therapy might be suitable, and provide examples of situations where human germline gene editing may be ethically permissible. Using such examples, we propose criteria to guide researchers and clinicians in deciding whether or not to pursue gene therapy as a treatment. Finally, we summarize how current progress in the field adheres to principles of biomedical ethics and highlight how this approach might fall short of ethical rigour using examples in the bioethics literature. Ultimately by addressing both the technical and ethical aspects of gene therapy and editing, new frameworks can be developed for the fair application of these potentially life-saving treatments.
-
DeepCRISTL: deep transfer learning to predict CRISPR/Cas9 functional and endogenous on-target editing efficiency.
MOTIVATION: CRISPR/Cas9 technology has been revolutionizing the field of gene editing in recent years. Guide RNAs (gRNAs) enable Cas9 proteins to target specific genomic loci for editing. However, editing efficiency varies between gRNAs. Thus, computational methods were developed to predict editing efficiency for any gRNA of interest. High-throughput datasets of Cas9 editing efficiencies were produced to train machine-learning models to predict editing efficiency. However, these high-throughput datasets have low correlation with functional and endogenous editing. Another difficulty arises from the fact that functional and endogenous editing efficiency is more difficult to measure, and as a result, functional and endogenous datasets are too small to train accurate machine-learning models on. RESULTS: We developed DeepCRISTL, a deep-learning model to predict the on-target efficiency given a gRNA sequence. DeepCRISTL takes advantage of high-throughput datasets to learn general patterns of gRNA on-target editing efficiency, and then uses transfer learning (TL) to fine-tune the model and fit it to the functional and endogenous prediction task. We pre-trained the DeepCRISTL model on more than 150 000 gRNAs, produced through the DeepHF study as a high-throughput dataset of three Cas9 enzymes. We improved the DeepHF model by multi-task and ensemble techniques and achieved state-of-the-art results over each of the three enzymes: up to 0.89 in Spearman correlation between predicted and measured on-target efficiencies. To fine-tune model weights to predict on-target efficiency of functional or endogenous datasets, we tested several TL approaches, with gradual learning being the overall best performer, both when pre-trained on DeepHF and when pre-trained on CRISPROn, another high-throughput dataset. DeepCRISTL outperformed state-of-the-art methods on all functional and endogenous datasets. Using saliency maps, we identified and compared the important features learned by the model in each dataset. We believe DeepCRISTL will improve prediction performance in many other CRISPR/Cas9 editing contexts by leveraging TL to utilize both high-throughput datasets, and smaller and more biologically relevant datasets, such as functional and endogenous datasets. AVAILABILITY AND IMPLEMENTATION: DeepCRISTL is available via github.com/OrensteinLab/DeepCRISTL. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
-
CRISPR-Cas Gene Perturbation and Editing in Human Induced Pluripotent Stem Cells.
Directing the fates of human pluripotent stem cells (hPSC) to generate a multitude of differentiated cell types allows the study of the genetic regulation of human development and disease. The translational potential of hPSC is maximized by exploiting CRISPR to silence or activate genes with spatial and temporal precision permanently or reversibly. Here, we summarize the increasingly refined and diverse CRISPR toolkit for the latter forms of gene perturbation in hPSC and their downstream applications. We discuss newer methods to install edits efficiently with single nucleotide resolution and describe pooled CRISPR screens as a powerful means of unbiased discovery of genes associated with a phenotype of interest. Last, we discuss the potential of these combined technologies in the treatment of hitherto intractable human diseases and the challenges to their implementation in the clinic.
-
Protocol for Prime Editing with Engineered PAM-Flexible Cas9 Variants.
Prime editing (PE) is a precision genome-editing technology that allows all types of editing, such as substitutions, insertions, and deletions, at the target site in the genome of a cell without double-strand breaks (DSBs) or donor DNA. However, the targeting range of existing prime editing systems is limited due to their dependence on the NGG protospacer adjacent motif (PAM) recognized by Streptococcus pyogenes Cas9 (spCas9). To address this limitation, we have engineered prime editor variants incorporating PAM-flexible Cas9 proteins. These variants significantly expand the range of targetable genomic sites, allowing for the introduction of mutations previously inaccessible to standard editing methods. Here, we provide detailed methods for applying these PAM-flexible PE variants in human cell lines.
-
Gene editing as a therapeutic strategy for spinocerebellar ataxia type-3.
Spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph disease, is a neurodegenerative disease caused by expanded polyglutamine repeats in exon 10 of the ataxin-3 gene, ATXN3. The accumulation of mutant ATXN3 protein leads to severe clinical manifestations and premature death. Clinically, SCA3 pathology is characterized by progressive ataxia leading to motor incoordination that may affect balance, gait and speech, and neuropathologically by a progressive degeneration of the spinal cord and cerebellum, as well as the cerebral cortex and basal ganglia. Although SCA3 is a rare disease, it is the most common autosomal dominant spinocerebellar ataxia worldwide. Its geographical distribution varies worldwide, with peak prevalence in certain regions of Brazil, Portugal and China. In 1994, the identification of the polyglutamine expansion in the ATXN3 gene made it possible not only to diagnose this pathology but also to dissect the mechanisms leading to cellular degeneration. As a monogenic disease for which only symptomatic treatment is available, the ATXN3 gene represents an attractive therapeutic target for gene editing strategies.
-
Biofortification of tomatoes with beta-carotene through targeted gene editing.
Vitamin A deficiency is one of the most severe micronutrient-related health issues worldwide. Tomatoes, a widely cultivated crop for their adaptability, nutritional value, and lycopene content (a beta-carotene precursor), are ideal candidates for biofortification. In this study, CRISPR-mediated knockout mutants (cr-SlLCYe and cr-SlBCH) were generated to enhance the precursor supply to the β-carotene biosynthetic pathway and reduce its degradation. Carotenoids profiling showed that β-carotene levels in the mutants were 1.7 to 2.5-fold higher than in the wild-type, whereas lycopene levels remained unaltered without altering lycopene content. To evaluate potential trade-offs, the characteristics of the mutant fruits were comprehensively assessed, including appearance quality (color, firmness), nutritional quality (sugars, organic acids, vitamin C), and postharvest traits (shelf life, resistance to Botrytis cinerea). These results provide a new strategy for elevating β-carotene without compromising fruit quality and offer new insights into combating vitamin A deficiency through targeted tomato breeding programs.
-
Repair-seq: Seeking and Perturbing DNA Repair.
-
CRISPR/Cas9 in plant biotechnology: applications and challenges.
The application of plant biotechnology to enhance beneficial traits in crops is now indispensable because of food insecurity due to increasing global population and climate change. The recent biotechnological development of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated system 9 (Cas9) allows for a more simple and precise method of gene editing, which is now preferred compared to Zinc Finger Nucleases (ZFNs) and Transcription Activator-like Effector Nucleases (TALENs). In this review, recent progress in utilizing CRISPR/Cas9-mediated gene editing in plants to enhance certain traits in beneficial crops, including rice, soybean, and oilseed rape, is discussed. In addition, novel methods of applying the CRISPR/Cas9 system in live cell imaging are also extensively reviewed. Despite all the applications, the existing delivery methods of CRISPR/Cas9 fail to provide consistent results and are inefficient for in planta transformation. Hence, research should be focused on improving current delivery methods or developing novel ones to facilitate CRISPR/Cas9-based gene editing studies. Strict regulations on the sale and commercial growth of gene-edited crops have restricted more efforts in applying CRISPR/Cas9 technology in plant species. Therefore, a shift in public viewpoint toward gene editing would help to propel scientific progress rapidly.
-
CRISPR/Cas9-mediated disruption of the immediate early-0 and 2 as a therapeutic approach to Bombyx mori nucleopolyhedrovirus in transgenic silkworm.
The CRISPR/Cas9 system is a powerful tool for the treatment of infectious diseases. In our previous study, we knocked out the Bombyx mori nucleopolyhedrovirus (BmNPV) key genes and BmNPV-dependent host factor to generate transgenic antiviral strains. To further expand the range of target genes for BmNPV and more effectively prevent and control pathogenic infections, we performed gene editing and antiviral analysis by constructing a target-directed baculovirus early transcriptional activator immediate early-0 (ie-0) and 2 (ie-2) transgenic silkworm line. We hybridized it with Cas9 transgenic line to produce a double-positive transgenic Cas9(+)/sgIE0-sgIE2(+) line that could activate the CRISPR gene editing system. We first demonstrated that the system is capable of efficiently editing target genes and resulting in fragment deletions in the BmNPV genome. Survival rate of the transgenic Cas9(+)/sgIE0-sgIE2(+) line reached 65% after inoculation with 1 × 106 occlusion bodies/larva. Molecular analysis showed that BmNPV DNA replication and viral gene expression level in the transgenic Cas9(+)/sgIE0-sgIE2(+) line were significantly inhibited compared with the control Cas9(-)/sgIE0-sgIE2(-) line. These results indicated that IE-0 and IE-2, as baculovirus early transcriptional activators, can be used as target sites for gene therapy and that multigene editing could expand the range of target sites for research to create silkworm resistance breeds.
-
Baculoviral delivery of CRISPR/Cas9 facilitates efficient genome editing in human cells.
The CRISPR/Cas9 system is a highly effective tool for genome editing. Key to robust genome editing is the efficient delivery of the CRISPR/Cas9 machinery. Viral delivery systems are efficient vehicles for the transduction of foreign genes but commonly used viral vectors suffer from a limited capacity in the genetic information they can carry. Baculovirus however is capable of carrying large exogenous DNA fragments. Here we investigate the use of baculoviral vectors as a delivery vehicle for CRISPR/Cas9 based genome-editing tools. We demonstrate transduction of a panel of cell lines with Cas9 and an sgRNA sequence, which results in efficient knockout of all four targeted subunits of the chromosomal passenger complex (CPC). We further show that introduction of a homology directed repair template into the same CRISPR/Cas9 baculovirus facilitates introduction of specific point mutations and endogenous gene tags. Tagging of the CPC recruitment factor Haspin with the fluorescent reporter YFP allowed us to study its native localization as well as recruitment to the cohesin subunit Pds5B.
PubMed отдаёт не больше 10 000 первых результатов — уточните запрос, чтобы увидеть остальные.