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Rapid and efficient generation of GFP-knocked-in Drosophila by the CRISPR-Cas9-mediated genome editing.
The CRISPR-Cas9 technology has been a powerful means to manipulate the genome in a wide range of organisms. A series of GFP knocked-in (GFPKI ) Drosophila strains have been generated through CRISPR-Cas9-induced double strand breaks coupled with homology-directed repairs in the presence of donor plasmids. They visualized specific cell types or intracellular structures in both fixed and live specimen. We provide a rapid and efficient strategy to identify KI lines. This method requires neither co-integration of a selection marker nor prior establishment of sgRNA-expressing transgenic lines. The injection of the mixture of a sgRNA/Cas9 expression plasmid and a donor plasmid into cleavage stage embryos efficiently generated multiple independent KI lines. A PCR-based selection allows to identify KI fly lines at the F1 generation (approximately 4 weeks after injection). These GFPKI strains have been deposited in the Kyoto Drosophila stock center, and made freely available to researchers at non-profit organizations. Thus, they will be useful resources for Drosophila research.
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Traceable Nano-Biohybrid Complexes by One-Step Synthesis as CRISPR-Chem Vectors for Neurodegenerative Diseases Synergistic Treatment.
Abnormal protein aggregations are essential pathological features of neurodegenerative diseases. Eliminating while inhibiting the regeneration of these protein aggregates is considered an effective treatment strategy. Herein, the CRISPR/Cas9 gene-editing tool is employed to inhibit the regeneration of disease-related proteins, while chemical drugs are applied to eliminate the proteins that are produced. To efficiently deliver CRISPR-chem drugs into brain lesions, traceable nano-biohybrid complexes (F-TBIO) are constructed by one-step synthesis and CRISPR/Cas9 plasmids (CF-TBIO) are loaded in a controllable manner. CF-TBIO can knock out the BACE1 gene and reduce the burden of amyloid-β, and thereby significantly improve the cognitive abilities of 2xTg-AD mice. In particular, by prolonging the dosing interval, the pathological damage and behavioral abilities of 2xTg-AD mice are still significantly improved. During the therapeutic process, CF-TBIO with a high relaxation rate provides accurate imaging signals in the complex brain physiological environment. The finding shows that CF-TBIO has great potential to serve as a CRISPR-chem drug-delivery platform for neurodegenerative diseases therapy.
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Potential benefits of gene editing for the future of poultry farming.
The chicken is an exemplar of efficient intensive animal agriculture and provides two valuable food products, chicken meat and eggs. Only aquaculture is better, by efficiency, but poultry is still top, by mass of animal protein produced as food in the global context. However this efficiency and intensive production comes with a number of challenges. Though the genetics of selective breeding have led to dramatic improvements in yield, efficiency and product quality, traits that relate to disease and welfare outcomes have not been so tractable. These two issues are major impacts to the industry in terms of production and in terms of public perception. Both transgenic technology and genome editing have clear potential for impact in these two important areas. The reproductive biology of birds requires techniques very specific to birds to achieve heritable (germline) edited traits. These are quite involved and, even though they are now well-defined and reliable, there is room for improvement and advances can be expected in the future. Currently the key targets for this technology are modifying chicken genes involved in virus-receptor interactions and cellular response involved in infection. For the egg industry the technology is being applied to the issue of sex-selection for layer hens (and the removal of males), removal of allergens from egg white and the tailoring of eggs system to enhance the yield of influenza vaccine doses. Regulation and trading of the animals generated, and resulting food products, will significantly impact the value and future development of genome editing for poultry.
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CRISPR/Cas9 genome editing through in planta transformation.
In this review, the application of CRISPR/Cas9 plant genome editing using alternative transformation methods is discussed. Genome editing by the CRISPR/Cas9 system is usually implemented via the generation of transgenic plants carrying Cas9 and sgRNA genes in the genome. Transgenic plants are usually developed by in vitro regeneration from single transformed cells, which requires using different in vitro culture-based methods. Despite their common application, these methods have some disadvantages and limitations. Thus, some methods of plant transformation that do not depend on in vitro regeneration have been developed. These methods are known as "in planta" transformation. The main focus of this review is the so-called floral dip in planta transformation method, although other approaches are also described. The main features of in planta transformation in the context of CRISPR/Cas9 genome editing are discussed. Furthermore, multiple ways to increase the effectiveness of this approach and to broaden its use in different plant species are considered.
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Jennifer A. Doudna and Emmanuelle Charpentier.
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Toehold-Based CRISPR-dCas9 Transcriptional Activation Platform for Spatiotemporally Controllable Gene Therapy in Tumor and Diabetic Mouse Models.
The CRISPR-Cas system has been extensively employed as a genome editing tool with the dCas9-based transcriptional activation system emerging as a particularly promising approach for gene editing in the treatment of diseases at the gene level. Nevertheless, the challenge of achieving effective spatiotemporal control of the transcriptional activation system of dCas9 has thus far restricted its broader application. In this study, we present an miRNA-responsive CRISPR-dCas9 transcriptional activation (mCTA) system. This system is capable of responding specifically to exogenous and endogenous miRNAs in mammalian cells and enables the specific imaging of miRNAs during neural development or in the deep tissues of mice. Furthermore, the replacement of downstream functional genes with DTA has been demonstrated to result in the effective apoptosis of tumor cells and inhibition of xenografted tumor growth in mice. Finally, in a diabetic mouse model, the m122CTA system was shown to reduce the blood glucose in diabetic mice via the activation of PDX-1 gene. Our work provides an effective platform for miRNA imaging and gene therapy via spatiotemporal control of gene regulation.
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Research advances CRISPR gene editing technology generated models in the study of epithelial ovarian carcinoma.
Epithelial ovarian carcinoma (EOC), the most lethal gynecologic cancer, is often diagnosed at advanced stages, which urge us to explore the novel therapeutic strategies. Mouse models have played a crucial role in elucidating the molecular mechanisms for the development ovarian cancer and its therapeutic strategies. However, there are still various challenges in modeling the genetic drivers of ovarian cancer in animal models. Here, we provided an overview of the research advances for the molecular mechanisms underlying EOC development, therapeutic strategies, the CRISPR genome editing technology and its generated EOC models. The review also comprehensively discussed the advantages and obstacles of CRISPR in generating EOC mouse models and the promising therapeutic approach by correcting the oncogenes of EOC through in vivo delivery of gene-edited components. The development of more precise animal models, along with a deeper understanding of EOC molecular mechanisms, will dramatically benefit the investigation and treatment of EOC.
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Efficient Genome Editing Using 'NanoMEDIC' AsCas12a-VLPs Produced with Pol II-Transcribed crRNA.
Virus-like particles (VLPs) are an attractive vehicle for the delivery of Cas nuclease and guide RNA ribonucleoprotein complexes (RNPs). Most VLPs are produced by packaging SpCas9 and its sgRNA, which is expressed from the RNA polymerase III (Pol III)-transcribed U6 promoter. VLPs assemble in the cytoplasm, but U6-driven sgRNA is localized in the nucleus, which hinders the efficient formation and packaging of RNPs into VLPs. In this study, using the nuclease packaging mechanism of 'NanoMEDIC' VLPs, we produced VLPs with AsCas12a and exploited its ability to process pre-crRNA. This allowed us to direct crRNA in the cytoplasm as part of a Pol II-driven transcript where AsCas12a excised mature crRNA, thus boosting RNP incorporation into VLPs. CMV-driven crRNA increased Venus and CCR5 transgene knockout levels in 293 cells from 30% to 50-90% and raised the level of endogenous CXCR4 knockout in Jurkat T cells from 1% to 20%. Changing a single crRNA to an array of three or six identical crRNAs improved CXCR4 knockout rates by up to 60-70%. Compared to SpCas9-VLPs, the editing efficiencies of AsCas12a-VLPs were higher, regardless of promoter usage. Thus, we showed that AsCas12a and CMV-driven crRNA could be efficiently packaged into VLPs and mediate high levels of gene editing. AsCas12a-VLPs are a new and promising tool for the delivery of RNPs into mammalian cells that will allow efficient target genome editing and may be useful for gene therapy applications.
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Fostering responsible research with genome editing technologies: a European perspective.
In this consensus paper resulting from a meeting that involved representatives from more than 20 European partners, we recommend the foundation of an expert group (European Steering Committee) to assess the potential benefits and draw-backs of genome editing (off-targets, mosaicisms, etc.), and to design risk matrices and scenarios for a responsible use of this promising technology. In addition, this European steering committee will contribute in promoting an open debate on societal aspects prior to a translation into national and international legislation.
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Applications of CRISPR/Cas as a Toolbox for Hepatitis B Virus Detection and Therapeutics.
Hepatitis B virus (HBV) infection remains a significant global health challenge, leading to chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (HCC). Covalently closed circular DNA (cccDNA) and integrated HBV DNA are pivotal in maintaining viral persistence. Recent advances in CRISPR/Cas technology offer innovative strategies to inhibit HBV by directly targeting both cccDNA and integrated HBV DNA or indirectly by degrading HBV RNAs or targeting host proteins. This review provides a comprehensive overview of the latest advancements in using CRISPR/Cas to inhibit HBV, with a special highlight on newer non-double-strand (non-DSB) break approaches. Beyond the canonical use of CRISPR/Cas for target inhibition, we discuss additional applications, including HBV diagnosis and developing models to understand cccDNA biology, highlighting the diverse use of this technology in the HBV field.
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