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Found: 26 168
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New models: Gene-editing boom means changing landscape for primate work.
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Two stones, one bird: co-targeting facilitates precise gene editing of non-selectable genes in Chlamydomonas.
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A Recombinant Targeted Bionanoparticle In Vitro Expressing a Gene-Editing Protein for Epidermal Growth Factor Receptor Mutant Lung Cancer.
Epidermal growth factor receptor (EGFR) mutation is a significant driving factor in the occurrence and progression of lung cancer. How to effectively block the abnormal expression of EGFR remains a key issue that urgently needs to be addressed, as it is of vital importance for the effective treatment of this cancer. Here, we report a recombinant targeted bionanoparticle that in vitro expresses Cas9 protein (tBioNP vitro-Cas9) for gene editing of EGFR mutant lung cancer. The nanoparticle was developed by transfection of four plasmids (Gag-Cas9, Gag, sgRNA, VSV-G Azi) into 293T cells to form a type of bionanoparticle and modifying it with a targeted polymer material (DBCO-PEG-FA), and it showed a cancer-targeted property, faster cancer cellular uptake, higher gene editing efficiency with lower off-target effects, and therapy efficacy in mice, indicating a translational prospect. In conclusion, the study provides a recombinant bionanoparticle in vitro expressing a Cas9 gene editing system and offers a potential strategy for gene therapy of EGFR mutant lung cancer.
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Chemical Modifications in Nucleic Acid Therapeutics.
Nucleic acid-based therapies (NATs) have become an increasingly prominent class of drugs due to the recent clinical successes made possible by nucleic acid chemical modifications. This class of therapies includes reagents that inhibit gene expression (antisense oligonucleotides (ASOs) or RNA interference (RNAi)), modulate gene structure (splice-shifting ASOs), increase protein expression (messenger RNA (mRNA)) or direct specific editing of the mammalian genome (CRISPR/Cas gene editing). Each of these technologies relies on specific combinations of chemically modified nucleic acids to increase drug efficacy, safety, and uptake efficiency in desired cell types. The knowledge gained from years of characterizing the biochemical properties of chemically modified oligonucleotides (ONs) combined with recent regulatory approvals will hopefully accelerate more NATs into the clinic to treat currently undruggable or ultrarare diseases. This review discusses the most employed chemical modifications in each of the aforementioned nucleic acid-based technologies and provides an overview of select publications that have demonstrated milestones and successes in improving ON efficacy and/or mitigating undesired off-target effects. Key innovations in chemical modifications that are expanding clinical capabilities are highlighted, casting a positive light on the future of nucleic acid medicine.
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EPO revokes Broad's CRISPR patent.
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Visualized Multigene Editing System for Aspergillus niger.
The resistance markers could ensure the entry of the CRISPR/Cas9 system into Aspergillus niger cells instead of gene editing. To increase the efficiency of positive colony screening on the primary transformation plates, we designed a visualized multigene editing system (VMS) via a unique tRNA-guide RNA (gRNA) array containing the gRNAs of a pigment gene albA and target genes. Disruption of albA produces white colonies, and the sequences of the endogenous tRNAAla, tRNAPhe, tRNAArg, tRNAIle, and tRNALeu enhance gRNA release. The disruption efficiencies of multigene were analyzed in the A. niger strain AG11 using ammA, amyA, prtT, kusA, and glaA as reporters. In white colonies on the primary transformation plates, the disruption rates of one-, two-, three-, four-, and five-target genes reached 89.2, 70.91, 50, 22.41, and 4.17%, respectively. The VMS developed here provides an effective method for screening homokaryotic multigene editing strains of A. niger.
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Induced Pluripotent Stem Cells (iPSCs) and Gene Therapy: A New Era for the Treatment of Neurological Diseases.
To date, gene therapy has employed viral vectors to deliver therapeutic genes. However, recent progress in molecular and cell biology has revolutionized the field of stem cells and gene therapy. A few years ago, clinical trials started using stem cell replacement therapy, and the induced pluripotent stem cells (iPSCs) technology combined with CRISPR-Cas9 gene editing has launched a new era in gene therapy for the treatment of neurological disorders. Here, we summarize the latest findings in this research field and discuss their clinical applications, emphasizing the relevance of recent studies in the development of innovative stem cell and gene editing therapeutic approaches. Even though tumorigenicity and immunogenicity are existing hurdles, we report how recent progress has tackled them, making engineered stem cell transplantation therapy a realistic option.
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Disruption of diphthamide synthesis genes and resulting toxin resistance as a robust technology for quantifying and optimizing CRISPR/Cas9-mediated gene editing.
We have devised an effective and robust method for the characterization of gene-editing events. The efficacy of editing-mediated mono- and bi-allelic gene inactivation and integration events is quantified based on colony counts. The combination of diphtheria toxin (DT) and puromycin (PM) selection enables analyses of 10,000-100,000 individual cells, assessing hundreds of clones with inactivated genes per experiment. Mono- and bi-allelic gene inactivation is differentiated by DT resistance, which occurs only upon bi-allelic inactivation. PM resistance indicates integration. The robustness and generalizability of the method were demonstrated by quantifying the frequency of gene inactivation and cassette integration under different editing approaches: CRISPR/Cas9-mediated complete inactivation was ~30-50-fold more frequent than cassette integration. Mono-allelic inactivation without integration occurred >100-fold more frequently than integration. Assessment of gRNA length confirmed 20mers to be most effective length for inactivation, while 16-18mers provided the highest overall integration efficacy. The overall efficacy was ~2-fold higher for CRISPR/Cas9 than for zinc-finger nuclease and was significantly increased upon modulation of non-homologous end joining or homology-directed repair. The frequencies and ratios of editing events were similar for two different DPH genes (independent of the target sequence or chromosomal location), which indicates that the optimization parameters identified with this method can be generalized.
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Minimal 2'-O-methyl phosphorothioate linkage modification pattern of synthetic guide RNAs for increased stability and efficient CRISPR-Cas9 gene editing avoiding cellular toxicity.
Since its initial application in mammalian cells, CRISPR-Cas9 has rapidly become a preferred method for genome engineering experiments. The Cas9 nuclease is targeted to genomic DNA using guide RNAs (gRNA), either as the native dual RNA system consisting of a DNA-targeting CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA), or as a chimeric single guide RNA (sgRNA). Entirely DNA-free CRISPR-Cas9 systems using either Cas9 protein or Cas9 mRNA and chemically synthesized gRNAs allow for transient expression of CRISPR-Cas9 components, thereby reducing the potential for off-targeting, which is a significant advantage in therapeutic applications. In addition, the use of synthetic gRNA allows for the incorporation of chemical modifications for enhanced properties including improved stability. Previous studies have demonstrated the utility of chemically modified gRNAs, but have focused on one pattern with multiple modifications in co-electroporation with Cas9 mRNA or multiple modifications and patterns with Cas9 plasmid lipid co-transfections. Here we present gene editing results using a series of chemically modified synthetic sgRNA molecules and chemically modified crRNA:tracrRNA molecules in both electroporation and lipid transfection assessing indel formation and/or phenotypic gene knockout. We show that while modifications are required for co-electroporation with Cas9 mRNA, some modification patterns of the gRNA are toxic to cells compared to the unmodified gRNA and most modification patterns do not significantly improve gene editing efficiency. We also present modification patterns of the gRNA that can modestly improve Cas9 gene editing efficiency when co-transfected with Cas9 mRNA or Cas9 protein (> 1.5-fold difference). These results indicate that for certain applications, including those relevant to primary cells, the incorporation of some, but not all chemical modification patterns on synthetic crRNA:tracrRNA or sgRNA can be beneficial to CRISPR-Cas9 gene editing.
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Cellular and molecular targets in β-Thalassemia: advances in iron Regulation, Erythropoiesis, and Gene-Based therapies.
β-Thalassemia is a prevalent autosomal recessive genetic disorder caused by mutations in the β-globin gene, leading to impaired hemoglobin production and chronic anemia. While traditional treatments such as regular blood transfusions, iron chelation, and supportive care have significantly improved patient survival, these methods remain palliative. The availability of curative options is limited, with allogeneic hematopoietic stem cell transplantation (HSCT) presenting risks due to donor constraints and procedure-related complications. Recent advances in the molecular understanding of β-thalassemia have opened the door to novel therapeutic strategies. Gene therapy approaches, including lentiviral vector-mediated β-globin gene addition and CRISPR/Cas9-mediated genome editing targeting the BCL11A enhancer, have shown promising results in clinical trials and have gained regulatory approval in several regions. Pharmacological interventions, such as luspatercept, which enhances erythroid maturation, and other fetal hemoglobin inducers, offer valuable alternatives for patients who are ineligible for gene-based therapies. This review explores the current landscape of β-thalassemia, including its epidemiology, pathophysiology, and clinical manifestations, while highlighting recent advances in treatment options. We critically evaluate the clinical data emerging from gene therapies and other innovative treatments, address the ongoing challenges, and outline future directions for enhancing patient outcomes through personalized and accessible care strategies.
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