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: 26 190
Страница 589 из 1000-
Enhanced Cas12a editing in mammalian cells and zebrafish.
Type V CRISPR-Cas12a systems provide an alternate nuclease platform to Cas9, with potential advantages for specific genome editing applications. Here we describe improvements to the Cas12a system that facilitate efficient targeted mutagenesis in mammalian cells and zebrafish embryos. We show that engineered variants of Cas12a with two different nuclear localization sequences (NLS) on the C terminus provide increased editing efficiency in mammalian cells. Additionally, we find that pre-crRNAs comprising a full-length direct repeat (full-DR-crRNA) sequence with specific stem-loop G-C base substitutions exhibit increased editing efficiencies compared with the standard mature crRNA framework. Finally, we demonstrate in zebrafish embryos that the improved LbCas12a and FnoCas12a nucleases in combination with these modified crRNAs display high mutagenesis efficiencies and low toxicity when delivered as ribonucleoprotein complexes at high concentration. Together, these results define a set of enhanced Cas12a components with broad utility in vertebrate systems.
-
Improved Base Editor for Efficiently Inducing Genetic Variations in Rice with CRISPR/Cas9-Guided Hyperactive hAID Mutant.
-
CRISPR-Cas9 Cytidine-Base-Editor Mediated Continuous In Vivo Evolution in Aspergillus nidulans.
Filamentous fungi are important cell factories for producing chemicals, organic acids, and enzymes. Although several genome editing tools are available for filamentous fungi, few effectively enable continuous evolution for rational engineering of complex phenotype. Here, we present CRISPR-Cas9 cytidine-base-editor (CBE) assisted in vivo evolution by continuously delivering a combinatorial sgRNA library to filamentous fungi. The method was validated by targeting core genes of 46 natural product biosynthetic gene clusters in Aspergillus nidulans NRRL 8112 to eliminate fungal toxins via six rounds of evolution. NGS analysis revealed the average C-to-T conversion rates in the first, third, and sixth rounds were 2.02%, 5.25%, and 9.34%, respectively. Metabolic profiles of the evolved mutants exhibited significant changes, allowing for the isolation of clean-background strains with enhanced production of an antifungal compound Echinocandin B. This study demonstrates that CBE-mediated in vivo evolution greatly facilitates the iterative refinement of complex morphogenetic traits in filamentous fungi.
-
A Phytophthora palmivora Extracellular Cystatin-Like Protease Inhibitor Targets Papain to Contribute to Virulence on Papaya.
Papaya fruits, stems, and leaves are rich in papain, a cysteine protease that has been shown to mediate plant defense against pathogens and insects. Yet the oomycete Phytophthora palmivora is a destructive pathogen that infects all parts of papaya plants, suggesting that it has evolved cysteine protease inhibitors to inhibit papain to enable successful infection. Out of five putative extracellular cystatin-like cysteine protease inhibitors (PpalEPICs) from P. palmivora transcriptomic sequence data, PpalEPIC8 appeared to be unique to P. palmivora and was highly induced during infection of papaya. Purified recombinant PpalEPIC8 strongly inhibited papain enzyme activity, suggesting that it is a functional cysteine protease inhibitor. Homozygous PpalEPIC8 mutants were generated using CRISPR/Cas9-mediated gene editing via Agrobacterium-mediated transformation (AMT). Increased papain sensitivity of in-vitro growth and reduced pathogenicity during infection of papaya fruits were observed for the mutants compared with the wild-type strain, suggesting that PpalEPIC8, indeed, plays a role in P. palmivora virulence by inhibiting papain. This study provided genetic evidence demonstrating that plant-pathogenic oomycetes secrete cystatins as important weapons to invade plants. It also established an effective gene-editing system for P. palmivora by the combined use of CRISPR/Cas9 and AMT, which is expected to be applicable to other oomycetes.
-
Cytosine base editor-mediated high-efficiency myostatin editing in Hu sheep.
The cytosine base editor (CBE) enables precise C-to-T substitution without inducing DNA double-strand breaks, which offering a promising tool for editing livestock genomes to enhance economically valuable traits. In this study, using Hu sheep, characterized by high reproductive performance but suboptimal meat production as the research subject, two CBE-editing sgRNAs (sgM1 and sgM2) targeting the negative regulator Myostatin (MSTN) gene were designed. The results revealed a 75% editing efficiency of sgM2 at the parthenogenetically activated embryonic level with no detectable off-target effects. Thirty-four zygotes from five Hu sheep microinjected with sgM2 and CBE mRNA mixtures were transferred into four Hu sheep recipient ewes, yielding four lambs with confirmed MSTN editing and no off-target activity. Statistical analysis of growth performance data revealed that MSTN-edited Hu sheep exhibited significantly (P < 0.05) higher body weights at 120-180 days, and significantly (P < 0.05) enlarged muscle fiber cross-sectional areas compared to wild-type controls. Edited Hu sheep displayed reduced MSTN protein expression, elevated p-AKT levels, and diminished p-ERK and p-p38 signaling. In conclusion, MSTN-edited Hu sheep were highly efficient generated using CBE, and further analysis demonstrate that MSTN editing activates the AKT pathway while suppressing MAPK signaling, leading to muscle fiber hypertrophy and accelerated growth, which provides technical methodologies and breeding materials for developing fast-growing, meat-type Hu sheep-germplasm.
-
An optimized prime editing system for efficient modification of the pig genome.
Prime editing (PE) is a recent gene editing technology that can mediate insertions or deletions and all twelve types of base-to-base conversions. However, its low efficiency hampers the application in creating novel breeds and biomedical models, especially in pigs and other important farm animals. Here, we demonstrate that the pig genome is editable using the PE system, but the editing efficiency was quite low as expected. Therefore, we aimed to enhance PE efficiency by modulating both exogenous PE tools and endogenous pathways in porcine embryonic fibroblasts (PEFs). First, we modified the pegRNA by extending the duplex length and mutating the fourth thymine in a continuous sequence of thymine bases to cytosine, which significantly enhanced PE efficiency by improving the expression of pegRNA and targeted cleavage. Then, we targeted SAMHD1, a deoxynucleoside triphosphate triphosphohydrolase (dNTPase) that impedes the reverse transcription process in retroviruses, and found that treatment with its inhibitor, cephalosporin C zinc salt (CPC), increased PE efficiency up to 29-fold (4-fold on average), presumably by improving the reverse transcription process of Moloney murine leukemia virus reverse transcriptase (M-MLV RT) in the PE system. Moreover, PE efficiency was obviously improved by treatment with a panel of histone deacetylase inhibitors (HDACis). Among the four HDACis tested, panobinostat was the most efficient, with an efficiency up to 122-fold (7-fold on average), partly due to the considerable HDACi-mediated increase in transgene expression. In addition, the synergistic use of the three strategies further enhanced PE efficiency in PEFs. Our study provides novel approaches for optimization of the PE system and broadens the application scope of PE in agriculture and biomedicine.
-
Advances in Engineered Virus-Like Particles for Genome Editing and Therapy.
Engineered virus-like particles (eVLPs) have emerged as a promising class of delivery systems for genome editing agents. By combining the efficient cellular entry of viral vectors with the safety advantages of nonviral platforms, eVLPs enable transient delivery of ribonucleoproteins such as Cas9, base editors, and prime editors. Successive design strategies, from rational engineering to directed optimization, have progressively improved particle assembly, cargo stability, and editing efficiency. In parallel, pseudotyping approaches have expanded the versatility of eVLPs for cell-type-specific targeting. Recent preclinical studies highlight their potential across diverse applications, ranging from monogenic disease models to complex disorders, and support their advancement toward clinical translation. This review summarizes the structural and production principles of eVLPs, surveys key advances in their development, and discusses therapeutic opportunities and future challenges for their application in genome editing.
-
Crispr-SGRU: Prediction of CRISPR/Cas9 Off-Target Activities with Mismatches and Indels Using Stacked BiGRU.
CRISPR/Cas9 is a popular genome editing technology, yet its clinical application is hindered by off-target effects. Many deep learning-based methods are available for off-target prediction. However, few can predict off-target activities with insertions or deletions (indels) between single guide RNA and DNA sequence pairs. Additionally, the analysis of off-target data is challenged due to a data imbalance issue. Moreover, the prediction accuracy and interpretability remain to be improved. Here, we introduce a deep learning-based framework, named Crispr-SGRU, to predict off-target activities with mismatches and indels. This model is based on Inception and stacked BiGRU. It adopts a dice loss function to solve the inherent imbalance issue. Experimental results show our model outperforms existing methods for off-target prediction in terms of accuracy and robustness. Finally, we study the interpretability of this model through Deep SHAP and teacher-student-based knowledge distillation, and find it can provide meaningful explanations for sequence patterns regarding off-target activity.
-
Genome-wide microhomologies enable precise template-free editing of biologically relevant deletion mutations.
The functional effect of a gene edit by designer nucleases depends on the DNA repair outcome at the targeted locus. While non-homologous end joining (NHEJ) repair results in various mutations, microhomology-mediated end joining (MMEJ) creates precise deletions based on the alignment of flanking microhomologies (µHs). Recently, the sequence context surrounding nuclease-induced double strand breaks (DSBs) has been shown to predict repair outcomes, for which µH plays an important role. Here, we survey naturally occurring human deletion variants and identify that 11 million or 57% are flanked by µHs, covering 88% of protein-coding genes. These biologically relevant mutations are candidates for precise creation in a template-free manner by MMEJ repair. Using CRISPR-Cas9 in human induced pluripotent stem cells (hiPSCs), we efficiently create pathogenic deletion mutations for demonstrable disease models with both gain- and loss-of-function phenotypes. We anticipate this dataset and gene editing strategy to enable functional genetic studies and drug screening.
-
A Comprehensive Toolbox for Genome Editing in Cultured Drosophila melanogaster Cells.
Custom genome editing has become an essential element of molecular biology. In particular, the generation of fusion constructs with epitope tags or fluorescent proteins at the genomic locus facilitates the analysis of protein expression, localization, and interaction partners at physiologic levels. Following up on our initial publication, we now describe a considerably simplified, more efficient, and readily scalable experimental workflow for PCR-based genome editing in cultured Drosophila melanogaster cells. Our analysis at the act5C locus suggests that PCR-based homology arms of 60 bp are sufficient to reach targeting efficiencies of up to 80% after selection; extension to 80 bp (PCR) or 500 bp (targeting vector) did not further improve the yield. We have expanded our targeting system to N-terminal epitope tags; this also allows the generation of cell populations with heterologous expression control of the tagged locus via the copper-inducible mtnDE promoter. We present detailed, quantitative data on editing efficiencies for several genomic loci that may serve as positive controls or benchmarks in other laboratories. While our first PCR-based editing approach offered only blasticidin-resistance for selection, we now introduce puromycin-resistance as a second, independent selection marker; it is thus possible to edit two loci (e.g., for coimmunoprecipitation) without marker removal. Finally, we describe a modified FLP recombinase expression plasmid that improves the efficiency of marker cassette FLP-out. In summary, our technique and reagents enable a flexible, robust, and cloning-free genome editing approach that can be parallelized for scale-up.
PubMed отдаёт не больше 10 000 первых результатов — уточните запрос, чтобы увидеть остальные.