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Found: 26 179
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Artificial Virus Delivers CRISPR-Cas9 System for Genome Editing of Cells in Mice.
CRISPR-Cas9 has emerged as a versatile genome-editing platform. However, due to the large size of the commonly used CRISPR-Cas9 system, its effective delivery has been a challenge and limits its utility for basic research and therapeutic applications. Herein, a multifunctional nucleus-targeting "core-shell" artificial virus (RRPHC) was constructed for the delivery of CRISPR-Cas9 system. The artificial virus could efficiently load with the CRISPR-Cas9 system, accelerate the endosomal escape, and promote the penetration into the nucleus without additional nuclear-localization signal, thus enabling targeted gene disruption. Notably, the artificial virus is more efficient than SuperFect, Lipofectamine 2000, and Lipofectamine 3000. When loaded with a CRISPR-Cas9 plasmid, it induced higher targeted gene disruption efficacy than that of Lipofectamine 3000. Furthermore, the artificial virus effectively targets the ovarian cancer via dual-receptor-mediated endocytosis and had minimum side effects. When loaded with the Cas9-hMTH1 system targeting MTH1 gene, RRPHC showed effective disruption of MTH1 in vivo. This strategy could be adapted for delivering CRISPR-Cas9 plasmid or other functional nucleic acids in vivo.
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Combining Conformational Profiling of GPCRs with CRISPR/Cas9 Gene Editing Approaches.
Ligand-biased signaling could have a significant impact on drug discovery programs. As such, many approaches to screening now target a larger section of the signaling responses downstream of an individual G protein-coupled receptor (GPCR). Biosensor-based platforms have been developed to capture signaling signatures. Despite the ability to use such signaling signatures, they may still be particular to an individual cell type and thus such platforms may not be portable from cell to cell, necessitating further cell-specific biosensor development. We have developed a complementary strategy based on capturing receptor-proximal conformational profiles using intra-molecular BRET-based sensors composed of a Renilla luciferase donor engineered into the carboxy-terminus and CCPGCC motifs which bind fluorescent hairpin biarsenical dyes engineered into different positions into the receptor primary structure. Here, we discuss how these experiments can be conducted and combined with CRISPR/Cas9 genome editing to assess specific G protein-dependent and -independent events.
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Detection of CRISPR/Cas9-Induced Genomic Fragment Deletions in Barley and Generation of Homozygous Edited Lines via Embryogenic Pollen Culture.
The CRISPR/Cas9 system from Streptococcus pyogenes is an increasingly popular tool for genome editing due to its ease of application. Here we demonstrate genomic DNA fragment removal using RNA directed Cas9 nuclease in barley. The high mutation frequency confirms the exceptional efficiency of the system and its suitability for generating loss-of-function mutant lines that may be used in functional genetics approaches to study endomembrane trafficking pathways and posttranslational protein modifications. The generation of doubled haploids from genome edited plants allows the recovery of true breeding lines that are instantly homozygous for the edited alleles.
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Precision Recovery After Spinal Cord Injury: Integrating CRISPR Technologies, AI-Driven Therapeutics, Single-Cell Omics, and System Neuroregeneration.
Spinal cord injury (SCI) remains one of the toughest obstacles in neuroscience and regenerative medicine due to both severe functional loss and limited healing ability. This article aims to provide a key integrative, mechanism-focused review of the molecular landscape of SCI and the new disruptive therapy technologies that are now evolving in the SCI arena. Our goal is to unify a fundamental pathophysiology of neuroinflammation, ferroptosis, glial scarring, and oxidative stress with the translation of precision treatment approaches driven by artificial intelligence (AI), CRISPR-mediated gene editing, and regenerative bioengineering. Drawing upon advances in single-cell omics, systems biology, and smart biomaterials, we will discuss the potential for reprogramming the spinal cord at multiple levels, from transcriptional programming to biomechanical scaffolds, to change the course from an irreversible degeneration toward a directed regenerative pathway. We will place special emphasis on using AI to improve diagnostic/prognostic and inferred responses, gene and cell therapies enabled by genomic editing, and bioelectronics capable of rehabilitating functional connectivity. Although many of the technologies described below are still in development, they are becoming increasingly disruptive capabilities of what it may mean to recover from an SCI. Instead of prescribing a particular therapeutic fix, we provide a future-looking synthesis of interrelated biological, computational, and bioengineering approaches that conjointly chart a course toward adaptive, personalized neuroregeneration. Our intent is to inspire a paradigm shift to resolve paralysis through precision recovery and to be grounded in a spirit of humility, rigor, and an interdisciplinary approach.
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Comparative analysis of genome editing systems, Cas9 and BE3, in silkworms.
The clustered regularly interspaced short palindromic repeats (CRISPR) system and the Cas9-derived proteins have been applied to genome editing in various organisms. Traditional Cas9 is typically used to knockout genes or specific DNA fragments based on the generation of double-stranded breaks, whereas nCas9 and dCas9 are fused with effectors to perform base pair transitions or epigenetic modification and regulation. However, this system has off-target effects and can cause genomic structure variations. Here, we comparatively analyzed Cas9 and BE3, an initial base editor based on the nCas9 fusion protein, in silkworms. Our results showed that base editing was superior to Cas9 in silkworm cultured cells. BE3 introduced accurate termination codons, whereas Cas9 did not. Moreover, Cas9 induced chromosome translocation, chromosome fragment repetition, and chromosome fragment deletion, with the deletion frequency reaching up to 4.29%. BE3 was not able to induce these changes in our study. Furthermore, Cas9-derived proteins blocked ribosome advance and mRNA transcription for 9 days, with a 9.40% repression effect by combining with double-stranded DNA when single guide RNAs were targeted in the coding region in silkworms. Overall, our findings established a strategy for choosing suitable editing tools for various applications in different organisms.
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Flexible TAM requirement of TnpB enables efficient single-nucleotide editing with expanded targeting scope.
TnpBs encoded by the IS200/IS605 family transposon are among the most abundant prokaryotic proteins from which type V CRISPR-Cas nucleases may have evolved. Since bacterial TnpBs can be programmed for RNA-guided dsDNA cleavage in the presence of a transposon-adjacent motif (TAM), these nucleases hold immense promise for genome editing. However, the activity and targeting specificity of TnpB in homology-directed gene editing remain unknown. Here we report that a thermophilic archaeal TnpB enables efficient gene editing in the natural host. Interestingly, the TnpB has different TAM requirements for eliciting cell death and for facilitating gene editing. By systematically characterizing TAM variants, we reveal that the TnpB recognizes a broad range of TAM sequences for gene editing including those that do not elicit apparent cell death. Importantly, TnpB shows a very high targeting specificity on targets flanked by a weak TAM. Taking advantage of this feature, we successfully leverage TnpB for efficient single-nucleotide editing with templated repair. The use of different weak TAM sequences not only facilitates more flexible gene editing with increased cell survival, but also greatly expands targeting scopes, and this strategy is probably applicable to diverse CRISPR-Cas systems.
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CRISPR/Cas9 Ribonucleoprotein Delivery Enhanced by Lipo-Xenopeptide Carriers and Homology-Directed Repair Modulators: Insights from Reporter Cell Lines.
CRISPR-Cas9 genome editing is a versatile platform for studying and treating various diseases. Homology-directed repair (HDR) with DNA donor templates serves as the primary pathway for gene correction in therapeutic applications, but its efficiency remains a significant challenge. This study investigates strategies to enhance gene correction efficiency using a T-shaped lipo-xenopeptide (XP)-based Cas9 RNP/ssDNA delivery system combined with various HDR enhancers. Nu7441, a known DNA-PKcs inhibitor, was found to be most effective in enhancing HDR-mediated gene correction. An over 10-fold increase in HDR efficiency was achieved by Nu7441 in HeLa-eGFPd2 cells, with a peak HDR efficiency of 53% at a 5 nM RNP concentration and up to 61% efficiency confirmed by Sanger sequencing. Surprisingly, the total gene editing efficiency including non-homologous end joining (NHEJ) was also improved. For example, Nu7441 boosted exon skipping via NHEJ-mediated splice site destruction by 30-fold in a DMD reporter cell model. Nu7441 modulated the cell cycle by reducing cells in the G1 phase and extending the S and G2/M phases without compromising cellular uptake or endosomal escape. The enhancement in genome editing by Nu7441 was widely applicable across several cell lines, several Cas9 RNP/ssDNA carriers (LAF-XPs), and also Cas9 mRNA/sgRNA/ssDNA polyplexes. These findings highlight a novel and counterintuitive role for Nu7441 as an enhancer of both HDR and total gene editing efficiency, presenting a promising strategy for Cas9 RNP-based gene therapy.
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Nanotechnology and CRISPR/Cas-Mediated Gene Therapy Strategies: Potential Role for Treating Genetic Disorders.
Gene therapy has made substantial progress in the treatment of the genetic diseases, focussing on the reduction of characteristics of recessive/dominant disorders, as well as various cancers. Extensive research has been conducted in the past few decades to investigate the application of nanotechnology and CRISPR/Cas technology in gene therapy. Nanotechnology due to attributes such has targeted drug delivery, controlled release, scalability and low toxicity has gained attention of the medical world. CRISPR/Cas9 system is considered as an impactful genome editing tool in the area of next-generation therapeutics and molecular diagnostics. CRISPR technology emphasises on gene editing, gene regulation modulation, and formulation of defined genetic changes. Its applications in treatment of the genetic disorders are extended beyond traditional therapies. These techniques are being explored as treatment of several genetic disorders including Duchenne muscular dystrophy, cystic fibrosis, Alzheimer's disease, Parkinson's disease, and Huntington disease. Despite considerable therapeutic potential of gene therapy, several obstacles must be addressed before it can be widely adopted in clinical practice, particularly in terms of ensuring safety and effectiveness. As research advances in this captivating field, these therapies will become the primary treatments and will have significant beneficial effects on the lives of patients with genetic disorders.
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Compositional Optimization of CRISPR/Cas9 Lipid Nanoparticles for Efficient Knockdown of Target Genes.
Efficient and safe delivery systems remain a major barrier to the clinical translation of CRISPR/Cas9 gene-editing technologies; among these, formulation optimization of lipid nanoparticles (LNPs) is a key approach to improve delivery performance. Here, we constructed an orthogonal formulation library of LNPs using the ionizable lipids 4A2C2C6-A8 and 4A2C2C8-A8 at varied molar ratios and screened for optimal compositions. We identified formulations that combined high editing efficiency with low cytotoxicity; in HeLa-Luc cells, the optimized formulation achieved >80% knockout of the luciferase reporter. Further physicochemical and functional investigations showed that LNPs with relatively high zeta potential, mean diameters near 200 nm, and appropriate internal hydrophobicity-when paired with superior cellular uptake and endosomal escape capabilities-synergistically enhanced delivery efficiency. Using the compositionally optimized LNP to codeliver Cas9 mRNA and an sgRNA targeting HSP47 (whose overexpression has been implicated in fibrosis), we achieved efficient protein-level knockdown of HSP47 in L929 cells. These results provide important guidance for formulation optimization of CRISPR/Cas9 LNPs and support their potential application in antifibrotic therapies.
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Efficient CRISPR-Cas9-Mediated Genome Editing of the Cane Toad (Rhinella marina).
Invasive species inflict major ecological, economic, and cultural harm worldwide, highlighting the urgent need for innovative control strategies. Genome editing offers exciting possibilities for targeted control methods for invasive species. Here, we demonstrate CRISPR-Cas9 genome editing in the cane toad (Rhinella marina), one of Australia's most notorious invasive species, by targeting the tyrosinase gene to produce albino phenotypes as visual markers for assessing editing efficiency. Microinjection of Cas9 protein and guide RNAs into one-cell zygotes resulted in 87.6% of mosaic larvae displaying nearly complete albinism, with 2.3% exhibiting complete albinism. For completely albino individuals, genomic analysis confirmed predominantly frameshift mutations or large deletions at the target site, with no wild-type alleles detected. Germline transmission rates reflected the extent of albinism in the mosaic adult, with maternal transmission approaching 100%. This first application of CRISPR-Cas9 in the Bufonidae family opens possibilities for exploring basic research questions and population control strategies.
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