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Found: 26 182
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A dual-specific CRISPR-Cas nanosystem for precision therapeutic editing of liver disorders.
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CRISPR screens are feasible in TP53 wild-type cells.
A recent study by Haapaniemi et al (2018) reported that intact p53 signaling hampers CRISPR-based functional genomic screens. Brown et al report good performance of genome-scale screens in TP53 wild-type cells and reiterate best practices for CRISPR screening.
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Multistage-Responsive Gene Editing to Sensitize Ion-Interference Enhanced Carbon Monoxide Gas Therapy.
As a promising therapeutic modality targeting cancer, gas therapy still faces critical challenges, especially in enhancing therapeutic efficacy and avoiding gas poisoning risks. Here, a pH/glutathione (GSH) dual stimuli-responsive CRISPR/Cas9 gene-editing nanoplatform combined with calcium-enhanced CO gas therapy for precise anticancer therapy, is established. In the tumor microenvironment (TME), the fast biodegradation of the CaCO3 layer via pH-induced hydrolyzation allows glucose oxidase (GOx) to catalyze glucose for H2 O2 production, which further reacts with manganese carbonyl (MnCO) and achieves the precise release of CO gas. Simultaneously, in situ Ca2+ overload from CaCO3 degradation disturbs mitochondrial Ca2+ homeostasis, resulting in Ca2+ -driven reactive oxygen species (ROS) formation and subsequent mitochondrial apoptosis signaling pathway activation. Subsequently, by GSH-induced cleavage of a disulfide bond, the released Cas9/sgRNA (RNP) can achieve nuclear factor E2-related factor 2 (Nrf2) gene ablation to sensitize gas therapy by interfering with ROS signaling. This therapeutic modality endows codelivery of CRISPR, ions, and gas with smart control features, which demonstrates great potential for future clinical applications in precise nanomedicine.
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Startle: A star homoplasy approach for CRISPR-Cas9 lineage tracing.
CRISPR-Cas9-based genome editing combined with single-cell sequencing enables the tracing of the history of cell divisions, or cellular lineage, in tissues and whole organisms. Although standard phylogenetic approaches may be applied to reconstruct cellular lineage trees from this data, the unique features of the CRISPR-Cas9 editing process motivate the development of specialized models that describe the evolution of CRISPR-Cas9-induced mutations. Here, we introduce the "star homoplasy" evolutionary model that constrains a phylogenetic character to mutate at most once along a lineage, capturing the "non-modifiability" property of CRISPR-Cas9 mutations. We derive a combinatorial characterization of star homoplasy phylogenies and use this characterization to develop an algorithm, "Startle", that computes a maximum parsimony star homoplasy phylogeny. We demonstrate that Startle infers more accurate phylogenies on simulated lineage tracing data compared with existing methods and finds parsimonious phylogenies with fewer metastatic migrations on lineage tracing data from mouse metastatic lung adenocarcinoma.
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Xenotransplantation in the Age of Genome Editing: Results From the Expert Report for the Federal Ethics Committee on Nonhuman Biotechnology With a Special Focus on Animal Ethics.
BACKGROUND: The Federal Ethics Committee on Non-Human Biotechnology (ECNH) of Switzerland is an independent expert committee appointed by the Federal Council and mandated to advise the federal authorities from an ethical perspective in the field of nonhuman biotechnology and gene technology. Due to recent developments in the field of xenotransplantation after the introduction of genome editing technologies, the ECNH has commissioned an expert report on the ethical questions of xenotransplantation with a focus on animal ethics. The subject of the inquiry is, in particular, if current developments in the field of xenotransplantation raise new questions regarding ethics in the nonhuman realm or if existing questions have to be re-examined and answered anew. METHODS: An interdisciplinary approach was applied to answer this question. Based on the latest empirical results from medicine and biotechnology, xenotransplantation is analyzed and evaluated with reference to the dignity of the creature (Würde der Kreatur)-which is defined in the Swiss Federal Constitution-and the dignity of animals (Tierwürde) that is stipulated in the Swiss Animal Welfare Act and the Federal Act on Non-Human Gene Technology, as well as contemporary positions in the ethics of the human-animal relationship. RESULTS: The report concludes that genome editing for xenotransplantation does not generate any qualitatively new ethical issues concerning ethics in the nonhuman realm. However, contemporary biotechnological developments must be taken as an opportunity to discuss existing ethical issues in an urgent and intensified manner, particularly regarding the significance of animals' moral standing. The lack of consideration of animal-related aspects and the neglect of current developments and the state of the art of animal ethics in the recent discussion about xenotransplantation is a scientific, ethical, and political issue because animals are most negatively affected by xenotransplantation. This is especially relevant because the contemporary state of the art in animal ethics tends to consider and protect animals more strongly than in the past.
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Identification of squalene epoxidase in triterpenes biosynthesis in Poria cocos by molecular docking and CRISPR-Cas9 gene editing.
BACKGROUND: Squalene epoxidase is one of the rate-limiting enzymes in the biosynthetic pathway of membrane sterols and triterpenoids. The enzyme catalyzes the formation of oxidized squalene, which is a common precursor of sterols and triterpenoids. RESULT: In this study, the squalene epoxidase gene (PcSE) was evaluated in Poria cocos. Molecular docking between PcSE and squalene was performed and the active amino acids were identified. The sgRNA were designed based on the active site residues. The effect on triterpene synthesis in P. cocos was consistent with the results from ultra-high-performance liquid chromatography-quadruplex time-of-flight-double mass spectrometry (UHPLC-QTOF-MS/MS) analysis. The results showed that deletion of PcSE inhibited triterpene synthesis. In vivo verification of PcSE function was performed using a PEG-mediated protoplast transformation approach. CONCLUSION: The findings from this study provide a foundation for further studies on heterologous biosynthesis of P. cocos secondary metabolites.
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Long-Term Evaluation of Retinal Morphology and Function in Rosa26-Cas9 Knock-In Mice.
The CRISPR/Cas9 system is a robust, efficient, and cost-effective gene editing tool widely adopted in translational studies of ocular diseases. However, in vivo CRISPR-based editing in animal models poses challenges such as the efficient delivery of the CRISPR components in viral vectors with limited packaging capacity and a Cas9-associated immune response. Using a germline Cas9-expressing mouse model would help to overcome these limitations. Here, we evaluated the long-term effects of SpCas9 expression on retinal morphology and function using Rosa26-Cas9 knock-in mice. We observed abundant SpCas9 expression in the RPE and retina of Rosa26-Cas9 mice using the real-time polymerase chain reaction (RT-PCR), Western blotting, and immunostaining. SD-OCT imaging and histological analysis of the RPE, retinal layers, and vasculature showed no apparent structural abnormalities in adult and aged Cas9 mice. Full-field electroretinogram of adult and aged Cas9 mice showed no long-term functional changes in the retinal tissues because of constitutive Cas9 expression. The current study showed that both the retina and RPE maintain their phenotypic and functional features in Cas9 knock-in mice, establishing this as an ideal animal model for developing therapeutics for retinal diseases.
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Generation of CRISPR-edited birch plants without DNA integration using Agrobacterium-mediated transformation technology.
CRISPR/Cas9 system has emerged as a powerful tool in genome editing; however, generation of CRISPR-edited DNA-free plants is still challenging. In this study, Betula platyphylla (birch) was used to build a method to generate CRISPR-edited plant without foreign DNA integration using Agrobacterium-mediated transformation (CPDAT method). This technique utilizes transient genetic transformation to introduce T-DNA coding gRNA and Cas9 into birch cells, and T-DNA will express to synthesize gRNA and Cas9 protein, which will form a complex to cleave the target DNA site. The genome may be mutated due to DNA repair, and these mutations will be preserved and accumulated not dependent on whether T-DNA is integrated into the genome or not. After transient transformation, birch plants were cut into explants to induce adventitious buds without antibiotic selection pressure. Each adventitious bud can be considered as an independent potentially CRISPR-edited line for mutation detection. CRISPR-edited birch plants without foreign DNA integration are further selected by screening CRISPR-edited lines without T-DNA integration. Among 65 randomly chosen independent lines, the mutation rate was 80.00% including 40.00% of lines with both alleles mutated. In addition, 5 lines out of 65 studied lines (7.69%) were CRISPR-edited birch plants without DNA integration. In conclusion, this innovative method presents a novel strategy for generating CRISPR-edited birch plants, thereby significantly enhancing the efficiency of generating common CRISPR-edited plants. These findings offer considerable potential to develop plant genome editing techniques further.
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Engineering dispersed mycelium morphology in Aspergillus niger for enhanced mycoprotein production via CRISPR/Cas9-mediated genome editing.
Filamentous fungi are widely utilized in industrial fermentation processes due to their high productivity, with mycelial morphology directly influencing fermentation broth viscosity and target product yield, which is a critical parameter for process optimization. Aspergillus niger, an FDA-approved safe filamentous fungus, typically forms tightly packed mycelial pellets in submerged cultures, which severely restricts its industrial application potential by limiting mass transfer efficiency. To address this challenge, CRISPR/Cas9 mediated genome editing coupled with fermentation optimization enhanced microbial protein production in A. niger. Endogenous α-1,3-glucan synthase genes (agsA, agsB) and galactosaminogalactan (GAG) synthase genes (sph3, uge3) were disrupted using CRISPR/Cas9, achieving complete dispersion of filamentous pellets in liquid media. This morphological engineering strategy resulted in a 77.52 % increase in biomass and 39.98 % enhancement in mycelial protein content compared to the wild-type strain (A. niger Li2). Transcriptomic analysis revealed that the engineered strain (A. niger AnΔABSU) exhibited upregulated transporter proteins (ABC transporters, MFS transporters, sugar transporters), accelerating nutrient uptake and energy metabolism; altered cell wall integrity pathways, including activation of the MAPK signaling cascade and increased sensitivity to cell wall stressors; enhanced amino acid biosynthesis, driven by upregulated gene expression in key metabolic pathways. Furthermore, response surface methodology (RSM) with Box-Behnken design optimized the fermentation medium, yielding 16.67 g/L biomass and 45.91 % protein content, representing 115.37 % and 67.01 % improvements over the unoptimized wild-type control. This study establishes a novel paradigm for constructing high-efficiency microbial protein cell factories via integrated morphological-engineering and fermentation optimization.
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2017 sneak peek: What the new year holds for science.
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