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CRISPR/Cas9-Based Gene Editing in Soybean.
CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat/CRISPR-associated Cas9)-based gene editing is a robust tool for functional genomics research and breeding programs in various crops. In soybean (Glycine max), a number of laboratories have obtained mutants by the CRISPR/Cas9 system; however, there has not yet been a detailed method for the CRISPR/Cas9-based gene editing in soybean. Here, we describe the procedures for constructing the CRISPR/Cas9 plasmid suitable for soybean gene editing and the modified protocols for Agrobacterium-mediated soybean transformation and regeneration from cotyledonary node explants containing the Cas9/sgRNA (single-guide RNA) transgenes.
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Engineering of CRISPR/Cas9-mediated potyvirus resistance in transgene-free Arabidopsis plants.
Members of the eukaryotic translation initiation factor (eIF) gene family, including eIF4E and its paralogue eIF(iso)4E, have previously been identified as recessive resistance alleles against various potyviruses in a range of different hosts. However, the identification and introgression of these alleles into important crop species is often limited. In this study, we utilise CRISPR/Cas9 technology to introduce sequence-specific deleterious point mutations at the eIF(iso)4E locus in Arabidopsis thaliana to successfully engineer complete resistance to Turnip mosaic virus (TuMV), a major pathogen in field-grown vegetable crops. By segregating the induced mutation from the CRISPR/Cas9 transgene, we outline a framework for the production of heritable, homozygous mutations in the transgene-free T2 generation in self-pollinating species. Analysis of dry weights and flowering times for four independent T3 lines revealed no differences from wild-type plants under standard growth conditions, suggesting that homozygous mutations in eIF(iso)4E do not affect plant vigour. Thus, the established CRISPR/Cas9 technology provides a new approach for the generation of Potyvirus resistance alleles in important crops without the use of persistent transgenes.
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Transient editing catches the eye.
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Simplified CRISPR-Mediated DNA Editing in Multicellular Eukaryotes.
The CRISPR-Cas9 system is becoming an imperative tool to edit the genome of various organisms. The gene-editing study by the CRISPR-Cas9 system has revolutionized the diverse field of biomedical research, genome engineering, and gene therapy. CRISPR-Cas9 system has been modified to induce genome editing by small-guide RNAs, which function together with Cas9 nuclease for sequence-specific cleavage of target sequences. Here, we describe the simplified protocol of CRISPR-Cas9-mediated DNA editing in multicellular eukaryotes, including the construction of gRNA plasmids into vectors, screening of positive clones, transfections into 293FT cell line, and transduction into Jurkat cells. We also describe different bioinformatic tools to design suitable gRNAs with increased efficiency and decreased off-target events. Further, we describe the assessments of DNA editing by indel mutations and sequencing in transduced cells.
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Validating Single-Guide RNA for Aedes aegypti Gene Editing.
Creating transgenic mosquitoes allows for mechanistic studies of basic mosquito biology and the development of novel vector control strategies. CRISPR-Cas9 gene editing has revolutionized gene editing, including in mosquitoes. This protocol details part of the gene editing process of Aedes aegypti mosquitoes via CRISPR-Cas9, through testing and validating single-guide RNAs (sgRNAs). Gene editing activity varies depending on the sequence of sgRNAs used, so validation of sgRNA activity should be done before large-scale generation of mutants or transgenics. sgRNA is designed using online tools and synthesized in <1 h. Once mutants or transgenics are generated via embryo microinjection, sgRNA activity is validated by quick genotyping polymerase chain reaction (PCR) and DNA sequencing.
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UK first to approve CRISPR treatment for diseases: what you need to know.
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Compartment-Specific Biosensors Reveal a Complementary Subcellular Distribution of Bioactive Furin and PC7.
Furin trafficking, and that of related proprotein convertases (PCs), may regulate which substrates are accessible for endoproteolysis, but tools to directly test this hypothesis have been lacking. Here, we develop targeted biosensors that indicate Furin activity in endosomes is 10-fold less inhibited by decanoyl-RVKR-chloromethylketone and enriched >3-fold in endosomes compared to the trans-Golgi network (TGN). Endogenous PC7, which resists this inhibitor, was active in distinct vesicles. Only overexpressed PC7 activity reached the cell surface, endosomes, and the TGN. A PLC motif in the cytosolic tail of PC7 was dispensable for endosomal activity, but it was specifically required for TGN recycling and to rescue proActivin-A cleavage in Furin-depleted B16F1 melanoma cells. In sharp contrast, PC7 complemented Furin in cleaving Notch1 independently of PLC-mediated TGN access. Our study provides a proof in principle that compartment-specific biosensors can be used to gain insight into the regulation of PC trafficking and to map the tropism of PC-specific inhibitors.
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Optical Control of a CRISPR/Cas9 System for Gene Editing by Using Photolabile crRNA.
Currently CRISPR/Cas9 is a widely used efficient tool for gene editing. Precise control over the CRISPR/Cas9 system with high temporal and spatial resolution is essential for studying gene regulation and editing. Here, we synthesized a novel light-controlled crRNA by coupling vitamin E and a photolabile linker at the 5' terminus to inactivate the CRISPR/Cas9 system. The vitamin E modification did not affect ribonucleoprotein (RNP) formation of Cas9/crRNA/tracrRNA complexes but did inhibit the association of RNP with the target DNA. Upon light irradiation, vitamin E-caged crRNA was successfully activated to achieve light-induced genome editing of vascular endothelial cell-growth factor A (VEGFA) in human cells through a T7E1 assay and Sanger sequencing as well as gene knockdown of EGFP expression in EGFP stably expressing cells. This new caging strategy for crRNA could provide new methods for spatiotemporal photoregulation of CRISPR/Cas9-mediated gene editing.
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CRISPR cures and cancer vaccines: researchers can help to shepherd them to market.
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Application of CRISPR/Cas9 gene editing technique in the study of cancer treatment.
In recent years, gene editing, especially that using clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9, has made great progress in the field of gene function. Rapid development of gene editing techniques has contributed to their significance in the field of medicine. Because the CRISPR/Cas9 gene editing tool is not only powerful but also has features such as strong specificity and high efficiency, it can accurately and rapidly screen the whole genome, facilitating the administration of gene therapy for specific diseases. In the field of tumor research, CRISPR/Cas9 can be used to edit genomes to explore the mechanisms of tumor occurrence, development, and metastasis. In these years, this system has been increasingly applied in tumor treatment research. CRISPR/Cas9 can be used to treat tumors by repairing mutations or knocking out specific genes. To date, numerous preliminary studies have been conducted on tumor treatment in related fields. CRISPR/Cas9 holds great promise for gene-level tumor treatment. Personalized and targeted therapy based on CRISPR/Cas9 will possibly shape the development of tumor therapy in the future. In this study, we review the findings of CRISPR/Cas9 for tumor treatment research to provide references for related future studies on the pathogenesis and clinical treatment of tumors.
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