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Improved Genome Editing in the Ascidian Ciona with CRISPR/Cas9 and TALEN.
The ascidian Ciona intestinalis type A (or Ciona robusta) is an important organism for elucidating the mechanisms that make the chordate body plan. CRISPR/Cas9 and TAL effector nuclease (TALEN) are widely used to quickly address genetic functions in Ciona. Our previously reported method of CRISPR/Cas9-mediated mutagenesis in this animal has inferior mutation rates compared to those of TALENs. We here describe an updated way to effectively mutate genes with CRISPR/Cas9 in Ciona. Although the construction of TALENs is much more laborious than that of CRISPR/Cas9, this technique is useful for tissue-specific knockouts that are not easy even by the optimized CRISPR/Cas9 method.
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Special Issue: Latest Research on Plant Genomics and Genome Editing.
Over the past ten years, plant science has undergone a remarkable transformation driven by the convergence of next-generation sequencing, increasingly sophisticated bioinformatics tools, and the rise of targeted genome editing platforms [...].
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Advances in the delivery of RNA therapeutics: from concept to clinical reality.
The rapid expansion of the available genomic data continues to greatly impact biomedical science and medicine. Fulfilling the clinical potential of genetic discoveries requires the development of therapeutics that can specifically modulate the expression of disease-relevant genes. RNA-based drugs, including short interfering RNAs and antisense oligonucleotides, are particularly promising examples of this newer class of biologics. For over two decades, researchers have been trying to overcome major challenges for utilizing such RNAs in a therapeutic context, including intracellular delivery, stability, and immune response activation. This research is finally beginning to bear fruit as the first RNA drugs gain FDA approval and more advance to the final phases of clinical trials. Furthermore, the recent advent of CRISPR, an RNA-guided gene-editing technology, as well as new strides in the delivery of messenger RNA transcribed in vitro, have triggered a major expansion of the RNA-therapeutics field. In this review, we discuss the challenges for clinical translation of RNA-based therapeutics, with an emphasis on recent advances in delivery technologies, and present an overview of the applications of RNA-based drugs for modulation of gene/protein expression and genome editing that are currently being investigated both in the laboratory as well as in the clinic.
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Reactive oxygen species responsive nanomotors for gene edited metabolic disruption and immunotherapy.
While gene-editing-based tumor therapy holds promise, conventional passive-diffusion vectors face limited penetration in dense solid tumors. Here, we developed a ROS-driven gene editing nanomotor (RDN@PL), which takes hemin as the core and encapsulates CRISPR/Cas9 plasmids targeting LDHA (A glycolysis key enzyme). In tumor microenvironments, RDN@PL consumes extracellular ROS to fuel self-diffusiophoresis, achieving higher intratumoral accumulation than passive particles. Upon internalization, heme oxygenase-1 (HO-1) degrades RDN@PL, releasing CO and plasmids. LDHA knockout suppresses glycolysis while CO elevates mitochondrial ROS, which triggers apoptosis by disrupting metabolism and enhancing immunity. Simultaneously, extracellular ROS depletion by non-internalized nanomotors reverses immunogenic cell death (ICD) inhibition, enhancing CD8+ T cell infiltration in tumor. The Janus nanomotor enables extracellular ROS scavenging and intracellular ROS increment via HO-1-responsive cargo release and gene editing. This multi-level intervention strategy demonstrates 93.9 % tumor growth suppression in solid tumor models, providing a blueprint for engineering intelligent nanovesicles in precision oncology.
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CRISPR-/Cas9-Mediated Precise and Efficient Genome Editing in Drosophila.
The CRISPR/Cas9 system provides the means to make precise and purposeful modifications to the genome via homology-directed repair (HDR). In Drosophila, a wide variety of tools provide flexibility to achieve these ends. Here, we detail a method to generate precise genome edits via HDR that is efficient and broadly applicable to any Drosophila stock or species. sgRNAs are first tested for their cleavage efficiency by injecting embryos with Cas9/sgRNA ribonucleoproteins using commercially available Cas9 protein. Using an empirically validated sgRNA, HDR is performed using a donor repair plasmid that carries two transformation markers. A fluorescent eye marker that can be seamlessly removed using PiggyBac transposase marks integration of the repair sequence. A counter-selection marker that produces small rough eyes via RNAi against eyes absent is used to screen against imprecise HDR events. Altogether, the enhancements implemented in this method expand the ease and scope of achieving precise CRISPR/Cas9 genome edits in Drosophila.
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Advances in genome editing: the technology of choice for precise and efficient β-thalassemia treatment.
Beta (β)-thalassemia is one of the most significant hemoglobinopathy worldwide. The high prevalence of the β-thalassemia carriers aggravates the disease burden for patients and national economies in the developing world. The survival of β-thalassemia patients solely relies on repeated transfusions, which eventually results into multi-organ damage. The fetal γ-globin genes are ordinarily silenced at birth and replaced by the adult β-globin genes. However, mutations that cause lifelong persistence of fetal γ-globin, ameliorate the debilitating effects of β-globin mutations. Therefore, therapeutically reactivating the fetal γ-globin gene is a prime focus of researchers. CRISPR/Cas9 is the most common approach to correct disease causative mutations or to enhance or disrupt the expression of proteins to mitigate the effects of the disease. CRISPR/cas9 and prime gene editing to correct mutations in hematopoietic stem cells of β-thalassemia patients has been considered a novel therapeutic approach for effective hemoglobin production. However, genome-editing technologies, along with all advantages, have shown some disadvantages due to either random insertions or deletions at the target site of edition or non-specific targeting in genome. Therefore, the focus of this review is to compare pros and cons of these editing technologies and to elaborate the retrospective scope of gene therapy for β-thalassemia patients.
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Generation of Human 293-F Suspension NGFR Knockout Cells Using CRISPR/Cas9 Coupled to Fluorescent Protein Expression.
Suspension cells derived from human embryonic kidney cells (HEK 293) are attractive cell lines for retroviral vector production in gene therapeutic development studies and applications. The low-affinity nerve growth factor receptor (NGFR) is a genetic marker frequently used as a reporter gene in transfer vectors to detect and enrich genetically modified cells. However, the HEK 293 cell line and its derivatives endogenously express the NGFR protein. To eradicate the high background NGFR expression in future retroviral vector packaging cells, we here employed the CRISPR/Cas9 system to generate human suspension 293-F NGFR knockout cells. The expression of a fluorescent protein coupled via a 2A peptide motif to the NGFR targeting Cas9 endonuclease enabled the simultaneous depletion of cells expressing Cas9 and remaining NGFR-positive cells. Thus, a pure population of NGFR-negative 293-F cells lacking persistent Cas9 expression was obtained in a simple and easily applicable procedure.
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Creating Large Chromosomal Deletions in Rice Using CRISPR/Cas9.
Engineered CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9) is an efficient and the most popularly used tool for genome engineering in eukaryotic organisms including plants, especially in crop plants. This system has been effectively used to introduce mutations in multiple genes simultaneously, create conditional alleles, and generate endogenously tagged proteins. CRISPR/Cas9 hence presents great value in basic and applied research for improving the performance of crop plants in various aspects such as increasing grain yields, improving nutritional content, and better combating biotic and abiotic stresses. Besides above applications, CRISPR/Cas9 system has been shown to be very effective in creating large chromosomal deletions in plants, which is useful for genetic analysis of chromosomal fragments, functional study of gene clusters in biological processes, and so on. Here, we present a protocol of creating large chromosomal deletions in rice using CRISPR/Cas9 system, including detailed information about single-guide RNA design, vector construction, plant transformation, and large deletion screening processes in rice.
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Treatment of congenital coagulopathies, from biologic to biotechnological drugs: The relevance of gene editing (CRISPR/Cas).
Congenital coagulopathies have, throughout the history of medicine, been a focus of scientific study and of great interest as they constitute an alteration of one of the most important and conserved pathways of evolution. The first therapeutic strategies developed to address them were aimed at restoring the blood components lost during hemorrhage by administering whole blood or plasma. Later on, the use of cryoprecipitates was a significant breakthrough as it made it possible to decrease the volumes of blood infused. In the 1970' and 80', clotting factor concentrates became the treatment and, from the 1990's to the present day, recombinant factors -with increasingly longer half-lives- have taken over as the treatment of choice for certain coagulopathies in a seamless yet momentous transition from biological to biotechnological drugs. The beginning of this century, however, saw the emergence of new advanced (gene and cell) treatments, which are currently transforming the therapeutic landscape. The possibility to use cells and viruses as well as specific or bispecific antibodies as medicines is likely to spark a revolution in the world of pharmacology where therapies will be individualized and have long-term effects. Specifically, attention is nowadays focused on the development of gene editing strategies, chiefly those based on CRISPR/Cas technology. Rare coagulopathies such as hemophilia A and B, or even ultra-rare ones such as factor V deficiency, could be among those deriving the greatest benefit from these new developments.
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Transformed bovine trophoblast stem cell lines, characterization, gene editing and secretion†.
Trophoblast stem cells (TSCs) serve as a critical model for understanding placental development, early embryo-maternal interactions, and pregnancy establishment in mammals. In cattle, the developing trophectoderm plays an essential role in conceptus elongation and secretion of factors necessary for maternal recognition of pregnancy. Building on previous work identifying signaling pathways regulating bovine TSC self-renewal and differentiation, we report the generation and characterization of transformed bovine TSC (bTSC) lines derived from blastocysts via lentiviral transduction of simian vacuolating virus 40 large T antigen. These rapidly proliferating TSC cell lines, maintained in the presence of Rho-associated protein kinase (ROCK) inhibition, retain key morphological and transcriptional characteristics of bovine TSCs. Upon transforming growth factor β-induced differentiation, they exhibit morphological and molecular changes consistent with trophoblast maturation. To evaluate their utility for functional studies, we demonstrated stable gene introduction of tdTomato and EGFP using lentiviral vectors and employed CRISPR/Cas9-mediated gene editing to target lentiviral EGFP integration sites, confirming efficient gene deletion. Additionally, proteomic analysis of conditioned medium identified secreted proteins with potential roles in embryo-uterine interactions, aligning with factors previously reported in bovine conceptus secretomes. These findings establish transformed bTSC lines as a valuable model for investigating bovine trophoblast biology, functional gene studies, and trophoblast-endometrial signaling. By providing a renewable in vitro system with stable proliferative capacity, these cell lines enable further exploration of the molecular mechanisms governing early pregnancy in cattle.
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