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Found: 26 179
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Precise homology-directed installation of large genomic edits in human cells with cleaving and nicking high-specificity Cas9 variants.
Homology-directed recombination (HDR) between donor constructs and acceptor genomic sequences cleaved by programmable nucleases, permits installing large genomic edits in mammalian cells in a precise fashion. Yet, next to precise gene knock-ins, programmable nucleases yield unintended genomic modifications resulting from non-homologous end-joining processes. Alternatively, in trans paired nicking (ITPN) involving tandem single-strand DNA breaks at target loci and exogenous donor constructs by CRISPR-Cas9 nickases, fosters seamless and scarless genome editing. In the present study, we identified high-specificity CRISPR-Cas9 nucleases capable of outperforming parental CRISPR-Cas9 nucleases in directing genome editing through homologous recombination (HR) and homology-mediated end joining (HMEJ) with donor constructs having regular and 'double-cut' designs, respectively. Additionally, we explored the ITPN principle by demonstrating its compatibility with orthogonal and high-specificity CRISPR-Cas9 nickases and, importantly, report that in human induced pluripotent stem cells (iPSCs), in contrast to high-specificity CRISPR-Cas9 nucleases, neither regular nor high-specificity CRISPR-Cas9 nickases activate P53 signaling, a DNA damage-sensing response linked to the emergence of gene-edited cells with tumor-associated mutations. Finally, experiments in human iPSCs revealed that differently from HR and HMEJ genome editing based on high-specificity CRISPR-Cas9 nucleases, ITPN involving high-specificity CRISPR-Cas9 nickases permits editing allelic sequences associated with essentiality and recurrence in the genome.
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Inhibiting the growth of melanoma cells via hTERT gene editing using CRISPR-dCas9-dnmt3a system.
CRISPR-Cas9 gene-editing technology has pushed the boundaries of genetic modification. The principle of this method is based on the purposeful defense system of DNA degradation and will be one of the most powerful instruments for gene editing shortly. The purpose of this study was to evaluate the capability of this approach to manage melanoma cells. The present study used EF1a-hsaCas9-U6-gRNA as a hybrid vector of sgRNA and Cas9 for the transfection of A-375 melanoma cells. Transfection efficiency was enhanced by examining the two concentrations of 4 and 8 µg/mL of hexadimethrine bromide (trade name Polybrene). The existence of Cas9 in transfected cells was detected by flow cytometry. The expression level of the metabisulfite-modified hTERT gene was measured by real-time PCR technique. The presence of telomerase in cells was determined by flow cytometry and western blotting analysis. The hTERT gene promoter methylation was also evaluated by HRM assay. Finally, the induction of apoptosis in transfected A375 cells was assessed using flow cytometry. The results showed that the presence of gRNA significantly increased the transfection efficiency (up to about 7.75 times higher). The hTERT expression levels in A-375 cells were significantly decreased at different concentrations of Polybrene (in a dose-dependent manner) and various amounts of transfection (P < 0.05). The expression of hTERT in basal cells was not significantly different from the group transfected without gRNA (P˃0.05) but was significantly higher than the group transfected with gRNA (P < 0.05). The results of flow cytometry and western blotting analysis showed a decrease in hTERT level compared to cells transfected without gRNA as well as basal cells. The methylation of hTERT gene promoter in the cells transfected with gRNA at a concentration of 80 μg/mL in the presence of both 4 μg/mL and 8 μg/mL of Polybrene was significantly increased compared to those transfected without sRNA (P < 0.05). The flow cytometry results indicated no significant difference in the induction of apoptosis in the transfected cells compared to the basal cells (P < 0.05). Evidence suggests that the designed CRISPR/Cas9 system reduces the expression of the hTERT gene and telomerase presence, thereby inhibiting the growth of melanoma cells.
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p38 MAPK inhibition safeguards genome integrity during gene editing of hematopoietic stem and progenitor cells.
Ex vivo genome editing of hematopoietic stem and progenitor cells (HSPCs) holds significant therapeutic potential but remains constrained by genotoxic risks associated with nuclease-induced DNA double-strand breaks, DNA donor template delivery and sensing, and proliferation-induced stress during ex vivo manipulation. These processes can lead to chromosomal instability, large on-target deletions, donor mis-integration, off-target events, and impaired long-term stem cell function, raising safety concerns for clinical translation. Here, we evaluate the impact of transient p38 MAPK inhibition on genomic integrity during clinically relevant CRISPR-Cas9 editing and show that this intervention attenuates ex vivo culture-associated stress without increasing detectable genotoxic outcomes. Comprehensive genotoxicity analyses, including quantification of large on-target deletions, adeno-associated viral vector mis-integration, and CAST-seq mapping of translocations, reveal no measurable differences in gene editing-associated structural alterations upon p38 MAPK inhibition, while micronuclei were significantly reduced. Importantly, long-term xenotransplantation followed by whole-exome sequencing shows that p38i-treated HSPCs display a reduced mutational burden without evidence of increased genomic alterations. Collectively, these findings identify transient p38 MAPK inhibition as a strategy to improve the quality and long-term fitness of gene-edited HSPCs without detectable adverse effects on the genome editing outcomes, supporting its further evaluation as a refinement to clinically relevant CRISPR-Cas9 editing workflows.
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Efficient Gene Transfer and Gene Editing in Sterlet (Acipenser ruthenus).
The sturgeon (Acipenseriformes) is an important farmed species because of its economical value. However, neither gene transfer nor gene editing techniques have been established in sturgeon for molecular breeding and gene functional study until now. In this study, we accomplished gene transfer and gene editing in sterlet (Acipenser ruthenus), which has the shortest sexual maturation period of sturgeons. The plasmid encoding enhanced green fluorescent protein (EGFP) was transferred into the embryos of sterlet at injection concentration of 100 ng/μL, under which condition high survival rate and gene transfer rate could be achieved. Subsequently, exogenous EGFP was efficiently disrupted by transcription activator-like effector nucleases (TALENs) or clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 nuclease/guide RNA (gRNA), with injection concentrations of 300 ng/μL TALENs, or 100 ng/μL Cas9 nuclease and 30 ng/μL gRNA, respectively, under which condition high survival rate and gene mutation rate could be achieved. Finally, the endogenous gene no tail in sterlet was successfully mutated by Cas9 nuclease/gRNA. We observed the CRISPR-induced no tail mutation, at a high efficiency with the mutant P0 embryos displaying the expected phenotype of bent spine and twisted tail.
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Developmental competence of porcine genome-edited zygotes.
Genome editing in pigs has tremendous practical applications for biomedicine. The advent of genome editing technology, with its use of site-specific nucleases-including ZFNs, TALENs, and the CRISPR/Cas9 system-has popularized targeted zygote genome editing via one-step microinjection in several mammalian species. Here, we review methods to optimize the developmental competence of genome-edited porcine embryos and strategies to improve the zygote genome-editing efficiency in pigs.
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Efficient generation of targeted large insertions by microinjection into two-cell-stage mouse embryos.
Rapid, efficient generation of knock-in mice with targeted large insertions remains a major hurdle in mouse genetics. Here, we describe two-cell homologous recombination (2C-HR)-CRISPR, a highly efficient gene-editing method based on introducing CRISPR reagents into embryos at the two-cell stage, which takes advantage of the open chromatin structure and the likely increase in homologous-recombination efficiency during the long G2 phase. Combining 2C-HR-CRISPR with a modified biotin-streptavidin approach to localize repair templates to target sites, we achieved a more-than-tenfold increase (up to 95%) in knock-in efficiency over standard methods. We targeted 20 endogenous genes expressed in blastocysts with fluorescent reporters and generated reporter mouse lines. We also generated triple-color blastocysts with all three lineages differentially labeled, as well as embryos carrying the two-component auxin-inducible degradation system for probing protein function. We suggest that 2C-HR-CRISPR is superior to random transgenesis or standard genome-editing protocols, because it ensures highly efficient insertions at endogenous loci and defined 'safe harbor' sites.
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National Academies relax stance on germline edits.
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Oligonucleotide-directed mutagenesis for precision gene editing.
Differences in gene sequences, many of which are single nucleotide polymorphisms, underlie some of the most important traits in plants. With humanity facing significant challenges to increase global agricultural productivity, there is an urgent need to accelerate the development of these traits in plants. oligonucleotide-directed mutagenesis (ODM), one of the many tools of Cibus' Rapid Trait Development System (RTDS(™) ) technology, offers a rapid, precise and non-transgenic breeding alternative for trait improvement in agriculture to address this urgent need. This review explores the application of ODM as a precision genome editing technology, with emphasis on using oligonucleotides to make targeted edits in plasmid, episomal and chromosomal DNA of bacterial, fungal, mammalian and plant systems. The process of employing ODM by way of RTDS technology has been improved in many ways by utilizing a fluorescence conversion system wherein a blue fluorescent protein (BFP) can be changed to a green fluorescent protein (GFP) by editing a single nucleotide of the BFP gene (CAC→TAC; H66 to Y66). For example, dependent on oligonucleotide length, applying oligonucleotide-mediated technology to target the BFP transgene in Arabidopsis thaliana protoplasts resulted in up to 0.05% precisely edited GFP loci. Here, the development of traits in commercially relevant plant varieties to improve crop performance by genome editing technologies such as ODM, and by extension RTDS, is reviewed.
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Highly efficient genome editing using oocyte-specific zcas9 transgenic zebrafish.
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Rational Design and Optimisation of CRISPR-Cas9 Delivery Systems for Targeted Genomic Transformation.
The CRISPR-Cas9 genome-editing technique offers a promising therapeutic strategy for genetic disorders, including neurodegenerative diseases like Alzheimer's disease (AD), characterised by inherited susceptibility and progressive cognitive decline, as well as other hallmarks such as amyloid beta (Aβ1-42) plaques and neurofibril tangles (NFTs). However, the blood-brain barrier (BBB) poses a significant challenge to the effectiveness of gene editing components in the affected brain region and impedes clinical translation. This comprehensive review compares various CRISPR-Cas9 delivery vectors, viral, nonviral and physical, with a focus on their efficacy in neurological diseases such as AD. Viral vectors viz., adeno-associated viruses (AAVs) and lentiviruses (LVs) demonstrate high transduction efficiency and BBB permeability. AAVs are preferred for their low immunogenicity, minimal toxicity, high neuronal tropism and episomal persistence, enabling sustained expression without insertional mutagenesis. LVs offer larger genetic payloads but raise concerns about genomic integration and potential oncogenesis, though integration-defective variants mitigate these risks. Nonviral vectors, including peptide and polymer-based nanoparticles, lipid nanoparticles (LNPs) and Inorganic carriers such as gold and silver nanoparticles, are less immunogenic and easier to handle but require further optimisation for in vivo BBB crossing and endosomal escape. Physical methods such as electroporation and microinjection are suitable for in vitro/ex vivo use, while novel CNS-targeted strategies, such as RVG-tagged particles, TfR-directed LNPs and engineered AAV variants, enhance brain penetration via receptor-mediated transcytosis. These preclinical studies show that these technologies can successfully edit genes and provide therapeutic benefits, including amyloid reduction and cognitive improvement in AD models. Yet off-target effects, immune responses and regulatory hurdles persist. Overall, continuous innovation in delivery vector design and safety profile-targeting strategies is crucial for advancing CRISPR-Cas9 towards clinical therapies for AD-based therapies and related neurological disorders.
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