• KSBB Journal
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• v.25 no.3
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• pp.215-222
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• 2010

Monogenic diseases are resulted from modifications in a single gene of human cells. Because their treatment with pharmacological medicine have a temporary effect, continuous nursing care and retreatment are required. Gene therapy, gene targeting and induced pluripotent stem cell (iPSC) are considered permanent treatment methods of them. In gene therapy, however, retroviral vectors that have potential toxicity caused by random insertion of harmful virus are used as vehicles for transferring genetic materials. On the other hand, gene targeting could replace and remove the modified gene though homologous recombination (HR) induced by site-specific endonucleases. This short review provides a brief overview on the recently tailored endonucleses with high selectivity for HR.

• Asian-Australasian Journal of Animal Sciences
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• v.27 no.3
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• pp.324-329
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• 2014

To facilitate the construction of genetically-modified pigs, we produced cloned embryos derived from porcine fibroblasts transfected with a pair of engineered zinc finger nuclease (ZFN) plasmids to create targeted mutations and enriched using a reporter plasmid system. The reporter expresses RFP and eGFP simultaneously when ZFN-mediated site-specific mutations occur. Thus, double positive cells ($RFP^+/eGFP^+$) were selected and used for somatic cell nuclear transfer. Two types of reporter based enrichment systems were used in this study; the cloned embryos derived from cells enriched using a magnetic sorting-based system showed better developmental competence than did those derived from cells enriched by flow cytometry. Mutated sequences, such as insertions, deletions, or substitutions, together with the wild-type sequence, were found in the cloned porcine blastocysts. Therefore, genetic mutations can be achieved in cloned porcine embryos reconstructed with ZFN-treated cells that were enriched by a reporter-based system.

• Asian-Australasian Journal of Animal Sciences
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• v.29 no.10
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• pp.1500-1507
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• 2016

Myostatin (MSTN) can negatively regulate the growth and development of skeletal muscle, and natural mutations can cause "double-muscling" trait in animals. In order to block the inhibiting effect of MSTN on muscle growth, we transferred zinc-finger nucleases (ZFN) which targeted sheep MSTN gene into cultured fibroblasts. Gene targeted colonies were isolated from transfected fibroblasts by serial dilution culture and screened by sequencing. Two colonies were identified with mono-allele mutation and one colony with bi-allelic deletion. Further, we introduced the MSTN-ZFN mRNA into sheep embryos by microinjection. Thirteen of thirty-seven parthenogenetic embryos were targeted by ZFN, with the efficiency of 35%. Our work established the technical foundation for generation of MSTN gene editing sheep by somatic cloning and microinjection ZFN into embryos.

• Journal of Animal Reproduction and Biotechnology
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• v.34 no.3
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• pp.148-156
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• 2019

The Transgenic livestock can be useful for the production of disease-resistant animals, pigs for xenotranplantation, animal bioreactor for therapeutic recombinant proteins and disease model animals. Previously, conventional methods without using artificial nuclease-dependent DNA cleavage system were used to produce such transgenic livestock, but their efficiency is known to be low. In the last decade, the development of artificial nucleases such as zinc-finger necleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regulatory interspaced short palindromic repeat (CRISPR)/Cas has led to more efficient production of knock-out and knock-in transgenic livestock. However, production of knock-in livestock is poor. In mouse, genetically modified mice are produced by coinjecting a pair of knock-in vector, which is a donor DNA, with a artificial nuclease in a pronuclear fertilized egg, but not in livestock. Gene targeting efficiency has been increased with the use of artificial nucleases, but the knock-in efficiency is still low in livestock. In many research now, somatic cell nuclear transfer (SCNT) methods used after selection of cell transfected with artificial nuclease for production of transgenic livestock. In particular, it is necessary to develop a system capable of producing transgenic livestock more efficiently by co-injection of artificial nuclease and knock-in vectors into fertilized eggs.

• Journal of Plant Biotechnology
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• v.42 no.3
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• pp.154-160
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• 2015

The plant breeding technology was developed with genetic engineering. Many researchers and breeders have turned from traditional breeding to molecular breeding. Genetically modified organisms (GMO) were developed via molecular breeding technology. Currently, molecular breeding technologies facilitate efficient plant breeding without introducing foreign genes, in virtue by of gene editing technology. Gene targeting (GT) via homologous recombination (HR) is one of the best gene editing methods available to modify specific DNA sequences in genomes. GT utilizes DNA repair pathways. Thus, DNA repair systems are controlled to enhance HR processing. Engineered sequence specific endonucleases were applied to improve GT efficiency. Engineered sequence specific endonucleases like the zinc finger nuclease (ZFN), TAL effector nuclease (TALEN), and CRISPR-Cas9 create DNA double-strand breaks (DSB) that can stimulate HR at a target site. RecQl4, Exo1 and Rad51 are effectors that enhance DSB repair via the HR pathway. This review focuses on recent developments in engineered sequence specific endonucleases and ways to improve the efficiency of GT via HR effectors in plants.

• Korean Journal of Agricultural Science
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• v.47 no.4
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• pp.903-920
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• 2020

Since its first demonstration as a practical genome editing tool in the early 2010s, the use of clustered regularly interspaced short palindromic repeat (CRISPR) along with the endonuclease Cas9 (CRISPR/Cas9) has become an essential choice for generating targeted mutations. Due to its relative simplicity and cost-effectiveness compared to other molecular scissors, i.e., zinc finger nuclease (ZFN) and transcription activator-like effector nuclease (TALEN), the CRISPR/Cas9 system has been shown to have a massive influence on genetic studies regardless of the biological kingdom. Although the system is in the process of being established, numerous protocols have already been released for the system and there have been various topics of CRISPR related papers published each year in ever-increasing manner. Here, we will briefly introduce CRISPR/Cas9 system and discuss the variants of the CRISPR system. Also, their applications to crop improvement will be dealt with mainly ornamental crops among horticultural crops other than Arabidopsis as a model plant. Finally, some issues on the barriers restraining the use of CRISPR system on floricultural crops, the prospect of CRISPR system as a DNA-free genome editing tool with efficient facilitators and finally, the future perspectives on the CRISPR system will be described.

• Molecules and Cells
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• v.38 no.6
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• pp.475-481
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• 2015

Programmable nucleases, which include zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and RNA-guided engineered nucleases (RGENs) repurposed from the type II clustered, regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) system are now widely used for genome editing in higher eukaryotic cells and whole organisms, revolutionising almost every discipline in biological research, medicine, and biotechnology. All of these nucleases, however, induce off-target mutations at sites homologous in sequence with on-target sites, limiting their utility in many applications including gene or cell therapy. In this review, we compare methods for detecting nuclease off-target mutations. We also review methods for profiling genome-wide off-target effects and discuss how to reduce or avoid off-target mutations.

• Journal of Animal Reproduction and Biotechnology
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• v.34 no.2
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• pp.65-69
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• 2019

Since the development of the first genetically-modified mouse, transgenic animals have been utilized for a wide range of industrial applications as well as basic research. To date, these transgenic animals have been used in functional genomics studies, disease models, and therapeutic protein production. Recent advances in genome modification techniques such zinc finger nuclease (ZFN), transcription activator-like effector nucleases (TALEN), and clustered regularly interspaced short palindromic repeats (CRIPSR)-Cas9, have led to rapid advancement in the generation of genome-tailored livestock, as well as experimental animals; however, the development of genome-edited poultry has shown considerably slower progress compared to that seen in mammals. Here, we will focus primarily on the technical strategies for production of transgenic and gene-edited chickens, and their potential for future applications.

• Journal of Embryo Transfer
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• v.30 no.4
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• pp.299-303
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• 2015

KO mice provide an excellent tool to determine roles of specific genes in biomedical filed. Traditionally, knockout mice were generated by homologous recombination in embryonic stem cells. Recently, engineered nucleases, such as zinc finger nuclease, transcription activator-like effector nuclease and clustered regularly interspaced short palindromic repeats (CRISPR), were used to produce knockout mice. This new technology is useful because of high efficiency and ability to generate biallelic mutation in founder mice. Until now, most of knockout mice produced using engineered nucleases were C57BL/6 strain. In the present study we used CRISPR-Cas9 system to generate knockout mice in FVB strain. We designed and synthesized single guide RNA (sgRNA) of CRISPR system for targeting gene, Abtb2. Mouse zygote were obtained from superovulated FVB female mice at 8-10 weeks of age. The sgRNA was injected into pronuclear of the mouse zygote with recombinant Cas9 protein. The microinjected zygotes were cultured for an additional day and only cleaved embryos were selected. The selected embryos were surgically transferred to oviduct of surrogate mother and offsprings were obtained. Genomic DNA were isolated from the offsprings and the target sequence was amplified using PCR. In T7E1 assay, 46.7% among the offsprings were founded as mutants. The PCR products were purified and sequences were analyzed. Most of the mutations were founded as deletion of few sequences at the target site, however, not identical among the each offspring. In conclusion, we found that CRISPR system is very efficient to generate knockout mice in FVB strain.