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A method of genetic transformation and gene editing of succulents without tissue culture.
Succulents, valued for their drought tolerance and ornamental appeal, are important in the floriculture market. However, only a handful of succulent species can be genetically transformed, making it difficult to improve these plants through genetic engineering. In this study, we adapted the recently developed cut-dip-budding (CDB) gene delivery system to transform three previously recalcitrant succulent varieties - the dicotyledonous Kalanchoe blossfeldiana and Crassula arborescens and the monocotyledonous Sansevieria trifasciata. Capitalizing on the robust ability of cut leaves to regenerate shoots, these plants were successfully transformed by directly infecting cut leaf segments with the Agrobacterium rhizogenes strain K599. The transformation efficiencies were approximately 74%, 5% and 3.9%-7.8%, respectively, for K. blossfeldiana and C. arborescens and S. trifasciata. Using this modified CDB method to deliver the CRISPR/Cas9 construct, gene editing efficiency in K. blossfeldiana at the PDS locus was approximately 70%. Our findings suggest that succulents with shoot regeneration ability from cut leaves can be genetically transformed using the CDB method, thus opening up an avenue for genetic engineering of these plants.
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Highly Efficient Cpf1-Mediated Gene Targeting in Mice Following High Concentration Pronuclear Injection.
Cpf1 has emerged as an alternative to the Cas9 RNA-guided nuclease. Here we show that gene targeting rates in mice using Cpf1 can meet, or even surpass, Cas9 targeting rates (approaching 100% targeting), but require higher concentrations of mRNA and guide. We also demonstrate that coinjecting two guides with close targeting sites can result in synergistic genomic cutting, even if one of the guides has minimal cutting activity.
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Introducing Emerging Science for the Clinician.
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Introduction.
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Knock-in editing: it functionally corrects!
In this issue of Blood, Hubbard et al provide important proof-of-concept data on the utility of using genome editing to knock-in a wild-type (WT) complementary DNA (cDNA) to functionally correct disease-causing mutations in a gene in primary human T cells.
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CRISPR/Cas genome editing in plants: mechanisms, applications, and overcoming bottlenecks.
The CRISPR/Cas systems have emerged as transformative tools for precisely manipulating plant genomes and enhancement. It has provided unparalleled applications from modifying the plant genomes to resistant enhancement. This review manuscript summarises the mechanism, application, and current challenges in the CRISPR/Cas genome editing technology. It addresses the molecular mechanisms of different Cas genes, elucidating their applications in various plants through crop improvement, disease resistance, and trait improvement. The advent of the CRISPR/Cas systems has enabled researchers to precisely modify plant genomes through gene knockouts, knock-ins, and gene expression modulation. Despite these successes, the CRISPR/Cas technology faces challenges, including off-target effects, Cas toxicity, and efficiency. In this manuscript, we also discuss these challenges and outline ongoing strategies employed to overcome these challenges, including the development of novel CRISPR/Cas variants with improved specificity and specific delivery methods for different plant species. The manuscript will conclude by addressing the future perspectives of the CRISPR/Cas technology in plants. Although this review manuscript is not conclusive, it aims to provide immense insights into the current state and future potential of CRISPR/Cas in sustainable and secure plant production.
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Genome-edited baby claim provokes international outcry.
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Efficient generation of a CYP3A4-T2A-luciferase knock-in HepaRG subclone and its optimized differentiation.
CRISPR/Cas9 has allowed development of better and easier-to-use ADME models than traditional methods by complete knockout or knock-in of genes. However, gene editing in HepaRG cells remains challenging because long-term monoclonal cultivation may alter their differentiation capacity to a large extent. Here, CRISPR/Cas9 was used to generate a CYP3A4-T2A-luciferase knock-in HepaRG subclone by Cas9-mediated homologous recombination and monoclonal cultivation. The knock-in HepaRG-#9 subclone retained a similar differentiation potential to wildtype HepaRG cells (HepaRG-WT). To further improve differentiation and expand the applications of knock-in HepaRG cells, two optimized differentiation procedures were evaluated by comparison with the standard differentiation procedure using the knock-in HepaRG-#9 subclone and HepaRG-WT. The results indicated that addition of forskolin (an adenylate cyclase activator) and SB431542 (a TGF-β pathway inhibitor) to the first optimized differentiation procedure led to better differentiation consequence in terms of not only the initiation time for differentiation and morphological characterization, but also the mRNA levels of hepatocyte-specific genes. These data may contribute to more extensive applications of genetically modified HepaRG cells in ADME studies.
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Gene drive: a faster route to plant improvement.
Gene drives for control of vector-borne diseases have been demonstrated in insects but remain challenging in plants. Theoretically, they could be transformative in speeding breeding programs and contributing to food security through providing novel weed control methods. Zhang et al. now report the possibility of implementing gene drive in plants for the first time.
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CRISPR/Cas technology: fueling the future of Biofuel production with sugarcane.
The objective of present review is to provide a scientific overview of sugarcane as a potential feedstock for biofuel and use of genome editing approach for improvement of industrial and agronomical traits in sugarcane. Sugarcane, a perennial tropical grass with a high biomass index, is a promising feedstock for bioethanol production, and its bagasse, rich in lignocellulosic material, serves as an ideal feedstock for producing second-generation bioethanol. To improve the conversion of sugarcane biomass into biofuels, developing varieties with improved biomass degradability and high biomass and sucrose content is essential. The complex genome architecture and earlier lack of sequence data hindered biotechnological advancements in sugarcane, but recent genome sequence updates offer new opportunities for sugarcane improvement. The first genetically modified sugarcane was developed in 1992 by Bower and Birch using microprojectile bombardment of embryogenic callus. Since then, transgenic techniques have rapidly evolved, leading to the advancement of genome editing technologies. Application of genome editing tools particularly CRISPR/Cas system has been successfully used in sugarcane for editing. Recently, multiple alleles of the magnesium chelatase and acetolactate synthase genes in sugarcane have been successfully edited through multiplexing. Additionally, CRISPR-edited sugarcane varieties with modified cell wall components and increased sucrose content for enhanced bioethanol production have been developed. At the end, the future of CRISPR-edited crops will depend on how well regulatory frameworks adapt to the rapidly evolving technology.
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