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Found: 26 157
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How genome editing could be used in the treatment of cardiovascular diseases.
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Gene Editing in the Ascidian Phallusia mammillata and Tail Nerve Cord Formation.
Functional approaches for studying embryonic development have greatly advanced thanks to the CRISPR-Cas9 gene editing technique. Previously practiced in just a few organisms, these knockout techniques are now widely applied. Here we describe simple techniques for applying the CRISPR-Cas9 system to study the development of the nerve cord in the ascidian Phallusia mammillata.
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Recombineering in Non-Model Bacteria.
The technology of recombineering, in vivo genetic engineering, was initially developed in Escherichia coli and uses bacteriophage-encoded homologous recombination proteins to efficiently recombine DNA at short homologies (35 to 50 nt). Because the technology is homology driven, genomic DNA can be modified precisely and independently of restriction site location. Recombineering uses linear DNA substrates that are introduced into the cell by electroporation; these can be PCR products, synthetic double-strand DNA (dsDNA), or single-strand DNA (ssDNA). Here we describe the applications, challenges, and factors affecting ssDNA and dsDNA recombineering in a variety of non-model bacteria, both Gram-negative and -positive, and recent breakthroughs in the field. We list different microbes in which the widely used phage λ Red and Rac RecET recombination systems have been used for in vivo genetic engineering. New homologous ssDNA and dsDNA recombineering systems isolated from non-model bacteria are also described. The Basic Protocol outlines a method for ssDNA recombineering in the non-model species of Shewanella. The Alternate Protocol describes the use of CRISPR/Cas as a counter-selection system in conjunction with recombineering to enhance recovery of recombinants. We provide additional background information, pertinent considerations for experimental design, and parameters critical for success. The design of ssDNA oligonucleotides (oligos) and various internet-based tools for oligo selection from genome sequences are also described, as is the use of oligo-mediated recombination. This simple form of genome editing uses only ssDNA oligo(s) and does not require an exogenous recombination system. The information presented here should help researchers identify a recombineering system suitable for their microbe(s) of interest. If no system has been characterized for a specific microbe, researchers can find guidance in developing a recombineering system from scratch. We provide a flowchart of decision-making paths for strategically applying annealase-dependent or oligo-mediated recombination in non-model and undomesticated bacteria. © 2022 Wiley Periodicals LLC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA. Basic Protocol: ssDNA recombineering in Shewanella species Alternate Protocol: ssDNA recombineering coupled to CRISPR/Cas9 in Shewanella species.
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Engineering Molecular Immunity Against Plant Viruses.
Genomic engineering has been used to precisely alter eukaryotic genomes at the single-base level for targeted gene editing, replacement, fusion, and mutagenesis, and plant viruses such as Tobacco rattle virus have been developed into efficient vectors for delivering genome-engineering reagents. In addition to altering the host genome, these methods can target pathogens to engineer molecular immunity. Indeed, recent studies have shown that clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) systems that target the genomes of DNA viruses can interfere with viral activity and limit viral symptoms in planta, demonstrating the utility of this system for engineering molecular immunity in plants. CRISPR/Cas9 can efficiently target single and multiple viral infections and confer plant immunity. Here, we discuss the use of site-specific nucleases to engineer molecular immunity against DNA and RNA viruses in plants. We also explore how to address the potential challenges encountered when producing plants with engineered resistance to single and mixed viral infections.
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[Progress in gene editing technologies for Saccharomyces cerevisiae].
Saccharomyces cerevisiae is one of the most important hosts in metabolic engineering. Advanced gene editing technology has been widely used in the design and construction of S. cerevisiae cell factories. With the rapid development of gene editing technology, early gene editing technologies based on recombinase and homologous recombination have been gradually replaced by new editing systems. In this review, the principle and application of gene editing technology in S. cerevisiae are summarized. Here, we first briefly describe the classical gene editing techniques of S. cerevisiae. Then elaborate the genome editing system of MegNs, ZFNs and TALENs based on endonuclease. The latest research progress is especially introduced and discussed, including the CRISPR/Cas system, multi-copy integration of heterologous metabolic pathways, and genome-scale gene editing. Finally, we envisage the application prospects and development directions of Saccharomyces cerevisiae gene editing technology.
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Expanding the cell quantity of CRISPR/Cas9 gene editing by continuous microfluidic electroporation chip.
CRISPR/Cas9-mediated gene editing offers promising and safe therapeutic options for a wide range of diseases. The technical difficulty of efficiently acquiring large quantities of gene-edited therapeutic cells in a short time period is now preventing the widespread clinical application of CRISPR/Cas9-mediated gene editing. Herein, a Large Volume Continuous Electroporation Chip (LaViE-Chip) has been developed to address the challenge of acquiring sufficient quantities of genetically edited cells for CRISPR/Cas9 gene editing. By connecting multiple relatively narrow microfluidic channels in parallel, a satisfactory balance between cell flow volume and electric field uniformity was achieved with two simple off-chip electrodes, which also isolated harmful effects around electrodes from target cells. Meanwhile, by carefully designing the curvature of the microfluidic channel, hydrodynamic controlled rotation of target cells has been realized to improve the transfection efficiency and cell viability. With these improvements, the LaViE-Chip realized 71.06 % electrotransfection efficiency, 84.3 % cell viability, and 107 cell/min cell processing speed. Moreover, the first successful incessant CRISPR gene editing by electroporation has been demonstrated, laying the technical foundation of therapeutic CRISPR gene editing.
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Gene editing technology: shaping international standards for health and food safety assurance.
The emergence of gene editing technologies like CRISPR-Cas9 has revolutionized health and food safety, necessitating robust international standards. This Science & Society examines how these advances have shaped global regulatory frameworks, ethical standards, and international collaborations, emphasizing the need for cohesive and ethical applications across various sectors.
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A Protocol for High-efficiency Transformation and Genome Editing in Elite Wheat Cultivars.
CRISPR-Cas9-based editing offers a simple yet efficient method for introducing targeted mutations in the genes of interest. Low efficiency of transformation and a lack of reproducibility in editing genes in elite wheat cultivars are the bottlenecks in improving simply inherited traits. We describe a high-efficiency transformation protocol for editing elite wheat cultivars using JD633-GRF4-GIF1 CRISPR vector. This vector reduces the regeneration time of transformants to less than 90 days and enables transformation of elite cultivars.
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Hope, despair and CRISPR - the race to save one woman's life.
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[Development of a CRISPR/Cpf1 gene editing system in silkworm Bombyx mori].
The CRISPR/Cas9 gene editing system has been widely used in basic research, gene therapy and genetic engineering due to its high efficiency, fast speed and convenience. Meanwhile, the discovery of novel CRISPR/Cas systems in the microbial community also accelerated the emergence of novel gene editing tools. CRISPR/Cpf1 is the second type (V type) CRISPR system that can edit mammalian genome. Compared with the CRISPR/Cas9, CRISPR/Cpf1 can use 5'T-PAM rich region to increase the genome coverage, and has many advantages, such as sticky end of cleavage site and less homologous recombination repair. Here we constructed three CRISPR/Cpf1 (AsCpf1, FnCpf1 and LbCpf1) expression vectors in silkworm cells. We selected a highly conserved BmHSP60 gene and an ATPase family BmATAD3A gene to design the target gRNA, and constructed gHSP60-266 and gATAD3A-346 knockout vectors. The efficiency for editing the target genes BmATAD3A and BmHSP60 by AsCpf1, FnCpf1 and LbCpf1 were analyzed by T7E1 analysis and T-clone sequencing. Moreover, the effects of target gene knockout by different gene editing systems on the protein translation of BmHSP60 and BmATAD3A were analyzed by Western blotting. We demonstrate the CRISPR/Cpf1 gene editing system developed in this study could effectively edit the silkworm genome, thus providing a novel method for silkworm gene function research, genetic engineering and genetic breeding.
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