• Development and Reproduction
• /
• v.21 no.3
• /
• pp.343-349
• /
• 2017

tWith the development of the third-generation gene scissors, CRISPR-Cas9, concerns are being raised about ethical and social repercussions of the new gene-editing technology. In this situation, this article explores the legislation and interpretation of the positive laws in South Korea. The BioAct does not specify and regulate 'gene editing' itself. However, assuming that genetic editing is used in the process of research and treatment, we can look to the specific details of the regulations for research on humans as well as gene therapy research in order to see how genetic editing is regulated under the BioAct. BioAct differentiates the regulation between (born) humans and embryos etc. and the regulation differ entirely in the manner and scope. Moreover, due to the fact that gene therapy products are regarded as drugs, they fall under different regulations. The Korean Pharmacopoeia Act put stringent sanctions on clinical trials for gene therapy products and the official Notification "Approval and Examination Regulations for Biological Products, etc." by Food and Drug Safety Administration may be applied to gene editing for gene therapy purposes.

• Journal of Life Science
• /
• v.29 no.5
• /
• pp.537-544
• /
• 2019

Genome editing technology employing gene scissors has generated interest in molecular breeding in various fields, and the development of the third-generation gene scissors of the clustered, regularly interspaced short palindromic repeat (CRISPR) system has accelerated the field of molecular breeding through genome editing. In this study, we analyzed the possibility of silkworm molecular breeding using gene scissors by genomic and phenotypic analysis after editing the biogenesis of lysosome-related organelles (BmBLOS) gene of Bakokjam using the CRISPR/Cas9 system. Three types of guide RNAs (gRNA) were synthesized based on the BmBLOS gene sequence of Bakokjam. Complexes of the prepared gRNA and Cas9 protein were formed and introduced into Bombyx mori BM-N cells by electroporation. Analysis of the gene editing efficiency by T7 endonuclease I analysis revealed that the B4N gRNA showed the best efficiency. The silkworm genome was edited by microinjecting the Cas9/B4N gRNA complex into silkworm early embryos and raising the silkworms after hatching. The hatching rate was as low as 18%, but the incidence of mutation was over 40%. In addition, phenotypic changes were observed in about 70% of the G0 generation silkworms. Sequence analysis showed that the BmBLOS gene appeared to be a heterozygote carrying the wild-type and mutation in most individuals, and the genotype of the BmBLOS gene was also different in all individuals. These results suggest that although the possibility of silkworm molecular breeding using the CRISPR/Cas9 system would be very high, continued research on breeding and screening methods will be necessary to improve gene editing efficiency and to obtain homozygotes.

• BMB Reports
• /
• v.45 no.12
• /
• pp.686-692
• /
• 2012

Phenotypic analysis of gene-specific knockout (KO) mice has revolutionized our understanding of in vivo gene functions. As the use of mouse embryonic stem (ES) cells is inevitable for conventional gene targeting, the generation of knockout mice remains a very time-consuming and expensive process. To accelerate the large-scale production and phenotype analyses of KO mice, international efforts have organized global consortia such as the International Knockout Mouse Consortium (IKMC) and International Mouse Phenotype Consortium (IMPC), and they are persistently expanding the KO mouse catalogue that is publicly available for the researches studying specific genes of interests in vivo. However, new technologies, adopting zinc-finger nucleases (ZFNs) or Transcription Activator-Like Effector (TALE) Nucleases (TALENs) to edit the mouse genome, are now emerging as valuable and effective shortcuts alternative for the conventional gene targeting using ES cells. Here, we introduce the recent achievement of IKMC, and evaluate the significance of ZFN/TALEN technology in mouse genetics.

• The Korean Society of Law and Medicine
• /
• v.23 no.1
• /
• pp.121-148
• /
• 2022

CRISPR-Cas9 is one of the gene-editing technologies that infinite potential. It may provide human beings with many benefits or cause unanticipated challenges. The governance as standards setting or regulation of gene-editing technologies can contribute to keeping a balance between scientific value and ethical commitments. Guaranteeing public participation provides an additional opportunity to think about ethical and moral considerations: For whose benefit the internationally discussed governance of gene-editing technologies is directed at? There is a doubt regarding whether the governance justifies scientific researchers' gene-editing research. Suppose that governance promotes the advancement of CRISPR-Cas9, it should also encourage greater research responsibility. If not, there may be tragedies brought about by the misconduct of researchers. Thus, the essential matter on the governance for the research of CRISPR-Cas9 is the researchers' responsibility.

• Biomedical Science Letters
• /
• v.26 no.2
• /
• pp.114-119
• /
• 2020

Of the various types of mice used for genome editing, conditional knock-out (cKO) mice serve as an important model for studying the function of genes. cKO mice can be produced using loxP knock-in (KI) mice in which loxP sequences (34 bp) are inserted on both sides of a specific region in the target gene. These mice can be used as KO mice that do not express a gene at a desired time or under a desired condition by cross-breeding with various Cre Tg mice. Genome editing has been recently made easy by the use of third-generation gene scissors, the CRISPR-Cas9 system. However, very few laboratories can produce mice for genome editing. Here we present a more efficient method for producing loxP KI mice. This method involves the use of an HDR vector as the target vector and ssODN as the donor DNA in order to induce homologous recombination for producing loxP KI mice. On injecting 20 ng/µL of ssODN, it was observed that the target exon was deleted or loxP was inserted on only one side. However, on injecting 10 ng/µL of the target HDR vector, the insertion of loxP was observed on both sides of the target region. In the first PCR, seven mice were identified to be loxP KI mice. The accuracy of their gene sequences was confirmed through Sanger sequencing. It is expected that the loxP KI mice produced in this study will serve as an important tool for identifying the function of the target gene.

• Plant Biotechnology Reports
• /
• v.5 no.1
• /
• pp.9-17
• /
• 2011

With the development of next-generation sequencing technology, ever-expanding databases of genetic information from various organisms are available to researchers. However, our ability to study the biological meaning of genetic information and to apply our genetic knowledge to produce genetically modified crops and animals is limited, largely due to the lack of molecular tools to manipulate genomes. Recently, targeted cleavage of the genome using engineered DNA scissors called zinc finger nucleases (ZFNs) has successfully supported the precise manipulation of genetic information in various cells, animals, and plants. In this review, we will discuss the development and applications of ZFN technology for genome engineering and highlight recent reports on its use in plants.

• Food Science and Industry
• /
• v.52 no.2
• /
• pp.171-187
• /
• 2019

Novel food materials can be produced based on biotechnology such as genetic recombination, microbial fermentation, and enzymatic engineering by utilizing living organisms such as animal, plant, and microorganism or by applying the enzymes isolated from them. Especially, exploration and development of novel prebiotics and probiotics attracted great attention worldwide in the food industry, of which the research and industrial trends in food biotechnology field are promoting the production of next generation sweeteners and proliferation of beneficial bacteria in gastrointestinal tract. Development and commercialization of novel food materials by domestic bioprocessing technology have been sluggish due to the GMO/LMO food safety issues. Meanwhile, the US and EU do not perceive badly about gene manipulation technology, and the research is most active in the fields of crops and GMMs, respectively. Genetic scissors, which are considered as next generation technology, are notable since foreign genes do not remain in final products.

• Journal of Life Science
• /
• v.30 no.8
• /
• pp.701-707
• /
• 2020

Gene editing technology using the clustered regularly interspaced short palindromic repeat (CRISPR) and the CRISPR associated protein (Cas)9 has been highly anticipated in developing breeding techniques. In this study, we discuss gene scissors as a tool for silkworm molecular breeding through analysis of Bombyx mori Kynurenine 3-Monooxygenase (BmKMO) gene editing using the CRISPR/Cas9 system and analysis of generational transmission through mutagenesis and selective crossing. The nucleotide sequence of the BmKMO gene was analyzed, and three guide RNAs (gRNAs) were prepared. Each synthesized gRNA was combined with Cas9 protein and then analyzed by T7 endonuclease I after introduction into the BM-N silkworm cell line. To edit the silkworm gene, K1P gRNA and Cas9 complexes were subsequently microinjected into the silkworm embryos; the hatching rate was 18% and the incidence of mutation was 60%. The gene mutation was verified in the heterozygous G0 generation, but no phenotypic change was observed. In homozygotes generated by self-crossing, a mutant phenotype was observed. These results suggest that silkworm molecular breeding using the CRISPR/Cas9 system is possible and could be an effective way of shortening the time required.

• Korean Journal of Food Science and Technology
• /
• v.37 no.2
• /
• pp.321-327
• /
• 2005

Staphylococcus aureus is spread worldwide and can result in food poisoning outbreaks. Among samples collected from soil, water, protected houses, packing houses, employees, strawberries, and leaves, and analyzed for S. aureus contamination, 16% samples 'showed S. aureus contamination, particularly on employees' hands, scissors, and strawberries. Examination of enterotoxins A, B, and C genes of S. aureus by PCR revealed sea and seb in 92 and 38% of total strains, respectively, whereas sec was not detected. In conclusion, implementation of Good Agricultural Practice is necessary for preventing food-borne diseases of staphylococcal origin, thereby ensuring the safety of farm-to-table products.

• Journal of Mushroom
• /
• v.20 no.3
• /
• pp.178-182
• /
• 2022

Despite the long history of mushroom use, studies examining the genetic function of mushrooms and the development of new varieties via bio-molecular methods are significantly lacking compared to those examining other organisms. However, owing to recent developments, attempts have been made to use a novel gene-editing technique involving CRISPR/Cas9 technology and genetic scissors in mushroom studies. In particular, research is actively being conducted to utilize ribonucleoprotein particles (RNPs) that can be genetically edited with high efficiency without foreign gene insertion for ease of selection. However, RNPs are too large for Cas9 protein to pass through the cell membrane of the protoplasmic reticulum. Furthermore, guide RNA is unstable and can be easily decomposed, which remarkably affects gene editing efficiency. In this study, nanoparticles were used to mitigate the shortcomings of RNP-based gene editing techniques and to obtain transformants stably. We used Lentinula edodes (shiitake mushroom) Sanjo705-13 monokaryon strain, which has been successfully used in previous genome editing experiments. To identify a suitable osmotic buffer for the isolation of protoplast, 0.6 M and 1.2 M sucrose, mannitol, sorbitol, and KCl were treated, respectively. In addition, with various nanoparticle-forming materials, experiments were conducted to confirm genome editing efficiency via the formation of nanoparticles with calcium phosphate (CaP), which can be bound to Cas9 protein without any additional amino acid modification. RNPs/NP complex was successfully formed and protected nuclease activity with nucleotide sequence specificity.