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Found: 26 182
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Gene Editing in Rat Embryonic Stem Cells to Produce In Vitro Models and In Vivo Reporters.
Rat embryonic stem cells (ESCs) offer the potential for sophisticated genome engineering in this valuable biomedical model species. However, germline transmission has been rare following conventional homologous recombination and clonal selection. Here, we used the CRISPR/Cas9 system to target genomic mutations and insertions. We first evaluated utility for directed mutagenesis and recovered clones with biallelic deletions in Lef1. Mutant cells exhibited reduced sensitivity to glycogen synthase kinase 3 inhibition during self-renewal. We then generated a non-disruptive knockin of dsRed at the Sox10 locus. Two clones produced germline chimeras. Comparative expression of dsRed and SOX10 validated the fidelity of the reporter. To illustrate utility, live imaging of dsRed in neonatal brain slices was employed to visualize oligodendrocyte lineage cells for patch-clamp recording. Overall, these results show that CRISPR/Cas9 gene editing technology in germline-competent rat ESCs is enabling for in vitro studies and for generating genetically modified rats.
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Advanced strategies for CRISPR/Cas9 delivery and applications in gene editing, therapy, and cancer detection using nanoparticles and nanocarriers.
Cancer remains one of the deadliest diseases, and is characterised by the uncontrolled growth of modified human cells. Unlike infectious diseases, cancer does not originate from foreign agents. Though a variety of diagnostic procedures are available; their cost-effectiveness and accessibility create significant hurdles. Non-specific cancer symptoms further complicate early detection, leading to belated recognition of certain cancer. The lack of reliable biomarkers hampers effective treatment, as chemotherapy, radiation therapy, and surgery often result in poor outcomes and high recurrence rates. Genetic and epigenetic mutations play a crucial role in cancer pathogenesis, necessitating the development of alternate treatment methods. The advent of CRISPR/Cas9 technology has transformed molecular biology and exhibits potential for gene modification and therapy in various cancer types. Nonetheless, obstacles such as safe transport, off-target consequences, and potency must be overcome before widespread clinical use. Notably, this review delves into the multifaceted landscape of cancer research, highlighting the pivotal role of nanoparticles in advancing CRISPR/Cas9-based cancer interventions. By addressing the challenges associated with cancer diagnosis and treatment, this integrated approach paves the way for innovative solutions and improved patient outcomes.
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One-step homozygosity in precise gene editing by an improved CRISPR/Cas9 system.
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Allele-specific suppression of pathogenic bestrophin-1 transcripts by CRISPR/Cas9-mediated genome editing.
BACKGROUND: Treating autosomal dominant gene mutations remains challenging, particularly when mutations convey a gain-of-function or a dominant-negative effect, as standard gene supplementation strategies often fail to counteract the pathogenic allele. METHODS: In this study, we employed human induced pluripotent stem cell-derived retinal pigment epithelium (hiPSC-RPE) to investigate allele-specific CRISPR/Cas9 genome editing as a potential treatment for Best disease (BD), an autosomal dominant macular dystrophy caused by over 250 distinct mutations in the bestrophin-1 (BEST1) gene. We designed and evaluated single guide RNAs (sgRNA) targeting three known BEST1 mutations (p.(R218C), p.(A243V), and p.(I295del)), assessing their impact on BD-associated hiPSC-RPE phenotypes and BEST1 channel function. Computationally predicted sgRNAs were rigorously tested for on-target efficiency, allele specificity and genome-wide off-target activities. RESULTS: We found that shortening sgRNA length improved specificity in some cases, while introducing an additional mismatch generally compromised editing efficiency. Notably, only one of the three mutations yielded an sgRNA with both high cleavage efficiency and undetectable off-target effects in hiPSC-RPE cells. We then explored the consequences of allele-specific editing on BEST1 expression and function in clonal BD hiPSC-RPE lines. Eliminating the mutant BEST1 transcript led to enhanced BEST1 localization, improved protein stability and restoration of anion transport function. CONCLUSIONS: Taken together, our findings support allele-specific gene editing as a viable therapeutic strategy for selected BEST1 mutations, while underscoring the necessity for rigorous testing of computationally designed sgRNAs, given their mutation- and context-dependent variability.
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Cas9-Mediated Gene-Editing in the Malaria Mosquito Anopheles stephensi by ReMOT Control.
Innovative tools are essential for advancing malaria control and depend on an understanding of molecular mechanisms governing transmission of malaria parasites by Anopheles mosquitoes. CRISPR/Cas9-based gene disruption is a powerful method to uncover underlying biology of vector-pathogen interactions and can itself form the basis of mosquito control strategies. However, embryo injection methods used to genetically manipulate mosquitoes (especially Anopheles) are difficult and inefficient, particularly for non-specialist laboratories. Here, we adapted the ReMOT Control (Receptor-mediated Ovary Transduction of Cargo) technique to deliver Cas9 ribonucleoprotein complex to adult mosquito ovaries, generating targeted and heritable mutations in the malaria vector Anopheles stephensi without injecting embryos. In Anopheles, ReMOT Control gene editing was as efficient as standard embryo injections. The application of ReMOT Control to Anopheles opens the power of CRISPR/Cas9 methods to malaria laboratories that lack the equipment or expertise to perform embryo injections and establishes the flexibility of ReMOT Control for diverse mosquito species.
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Increasing the efficiency of CRISPR/Cas9-mediated precise genome editing in rats by inhibiting NHEJ and using Cas9 protein.
Precise modifications such as site mutation, codon replacement, insertion or precise targeted deletion are needed for studies of accurate gene function. The CRISPR/Cas9 system has been proved as a powerful tool to generate gene knockout and knockin animals. But the homologous recombination (HR)-directed precise genetic modification mediated by CRISPR/Cas9 is relatively lower compared with nonhomologous end-joining (NHEJ) pathway and extremely expected to be improved. Here, in this study 2 strategies were used to increase the precise genetic modification in rats. Scr7, a DNA ligase IV inhibitor, first identified as an anti-cancer compound, and considered as a potential NHEJ inhibitor, was used to increase the HR-mediated precise genetic modification. Meanwhile, the Cas9 protein instead of mRNA was used to save the mRNA to protein translation step to improve the precise modification efficiency. The Fabp2 and Dbndd1 loci were selected to knockin Cre and CreER(T2), respectively. Our result showed that both Scr7 and Cas9 protein can increase the precise modification.
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Valproic Acid Thermally Destabilizes and Inhibits SpyCas9 Activity.
The clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 system plays an important role in prokaryotic adaptive immunity. Due to its capacity for sequence-specific gene editing, CRISPR-Cas9 has become one of the most important tools widely used for genome editing in molecular biotechnology. However, its clinical application is currently limited by unwanted mutations at off-target sites. Various strategies have been developed for precise control of Cas9 in order to reduce these off-target effects, including chemical-based approaches. From a chemical screening, I observed that valproic acid (VPA) binds to and destabilizes Streptococcus pyogenes Cas9 (SpyCas9) protein in vitro, as well as in cells, while within its therapeutical concentration range under conditions of hyperthermia as demonstrated. Conditions were generated either by an external heat bag or in combination with the photothermal therapeutic agent indocyanine green activated by a near-infrared laser. Use of other histone deacetylase inhibitors failed, suggesting a histone deacetylase inhibition-independent function of VPA. Thus, this finding provides an uncomplicated thermotherapeutical approach for timely regulation of the activity of the CRISPR-Cas9 system at desired locations.
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Targeted Deletion of an Entire Chromosome Using CRISPR/Cas9.
CRISPR/Cas9 genome editing can facilitate efficient deletion of genomic region, but it has not been used to delete an entire chromosome. Here, Adikusuma et al. show proof-of-concept for efficient CRISPR-mediated selective chromosome deletion by removing the centromere or shredding the chromosome arm in mouse embryonic stem cells and zygotes.
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Developing a RecT-assisted endogenous CRISPR/SzCas9 system for precise genome editing in Streptococcus zooepidemicus.
Streptococcus zooepidemicus is the most commonly utilized strain for industrial-scale hyaluronic acid (HA) production due to its capacity to produce high-quality HA. However, the lack of efficient genetic manipulation approaches has significantly hindered the industrial application of this species. In this study, we developed a RecT-assisted endogenous CRISPR/SzCas9 system to enable markerless gene deletion, gene substitution, stop codon insertion, and iterative editing in the industrially significant strain S. zooepidemicus for hyaluronic acid production. A variety of editing outcomes were achieved, including a 3186-bp deletion of the hylb gene, replacement of VHb, and insertion of stop codons into the sagA gene. Ultimately, we successfully developed an engineered strain, designated SD-3, which exhibited reduced toxicity and non-hemolytic properties. The hyaluronic acid production of SD-3 increased by 39.7%, reaching 10.8 g/L in a 5-L fermenter. This achievement represents the highest titer of high molecular weight hyaluronic acid to date, characterized by a uniform and high molecular weight of up to 2.8 × 106 Da.
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Chemically-inducible CRISPR/Cas9 circuits for ultra-high dynamic range gene perturbation.
CRISPR/Cas9 technologies provide unique capabilities for modeling disease and understanding gene-to-phenotype connections. In cultured cells, chemical-mediated control of Cas9 activity can limit off-target effects and enable mechanistic study of essential genes. However, widely-used Tet-On systems often show leaky Cas9 expression, leading to unintended edits, as well as weak activity upon induction. Leakiness can be problematic in the context of Cas9 nuclease activity, which may result in cumulative DNA damage and degradation of the target cell genome over time. To overcome these deficiencies, we have established transgenic platforms that minimize Cas9 functionality in the OFF-state along with maximized and uncompromised ON-state gene editing efficiency. By combining conditional destabilization and inhibition of Cas9, we have developed an all-in-one (one or multiple guide RNAs and Cas9) ultra-tight, Tet-inducible system with exceptional dynamic range (ON vs. OFF-state) across various cell lines and targets. As an alternative to Tet-mediated induction, we have created a Branaplam-regulated splice switch module for low-baseline and robust Cas9 activity control. Lastly, for circumstances where DNA damage needs to be avoided, we have constructed a dual-control, Tet-inducible CRISPRi module for tight and potent transcriptional silencing. This upgraded suite of inducible CRISPR systems has broad applications for numerous cell types and experimental conditions.
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