• International Journal of Industrial Entomology and Biomaterials
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• v.39 no.1
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• pp.14-21
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• 2019

Genome editing by CRISPR/Cas9, a third-generation gene scissor in molecular breeding at the genome level, is attracting much attention as one of the breeding techniques of the future. In this study, genetic and phenotypic analysis was used to examine the responsiveness of the Bakokjam variety of the silkworm Bombyx mori to molecular breeding using CRISPR/Cas9 in editing the white egg 2 (w-2) gene. The nucleotide sequence of the w-2 gene was analyzed and three different guide RNAs (gRNA) were prepared. The 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, W1N and W2P gRNA and Cas9 complexes were microinjected into silkworm embryos. Based on the results of microinjection, the hatching rate was 16-24% and the incidence of mutation was 33-37%. The gene mutation was verified in the heterozygous F1 generation, but no phenotypic change was observed. In F2 homozygotes generated by F1 self-crosses, a mutant phenotype was observed. These results suggest that silkworm molecular breeding using the CRISPR/Cas9 system is possible and will be a very effective way to shorten the time required than the traditional breeding process.

• International Journal of Industrial Entomology and Biomaterials
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• v.37 no.1
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• pp.21-28
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• 2018

CRISPR/Cas9 gene editing system is an efficient method to mutation in a sequence specific manner. Here we report the direct transfection of the Cas9 nuclease and gene specific guide RNA can be used in BM-N cell line derived from Bombyx mori ovarian tissue to enfeeble function of endogenous gene in vitro. We have used gene editing system to negative regulation components of major signaling cascade, the Toll pathway, which controls B. mori resistance to microbe infections, such as fungi and gram positive bacteria. We demonstrate that the $I{\kappa}B-like$ protein Cactus may controls the activation of transcription factors such as Rel A and Rel B. The direct transfection of Cas9 nuclease and Cactus-specific guide-RNA complex may be used in BM-N cells to disrupt the function of endogenous genes in vitro. A mutation frequency of 30-40% was observed in the transfected cells, and various mutations caused the target region. Moreover, RT-PCR analysis revealed that Cactus gene was down regulated after these mutations. More importantly, mutation of BmCactus stimulated expression of lysozyme, moricin, and lebocin genes. These results suggest that the CRISPR/Cas9 systems are expected to efficiently induce site-specific mutations and it was possible to produce antimicrobial peptide through the gene editing.

• Development and Reproduction
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• v.27 no.4
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• pp.205-211
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• 2023

INTS14/VWA9, a component of the integrator complex subunits, plays a pivotal role in regulating the fate of numerous nascent RNAs transcribed by RNA polymerase II, particularly in the biogenesis of small nuclear RNAs and enhancer RNAs. Despite its significance, a comprehensive mutation model for developmental research has been lacking. To address this gap, we aimed to investigate the expression patterns of INTS14 during zebrafish embryonic development. We generated ints14 mutant strains using the CRISPR/Cas9 system. We validated the gRNA activity by co-injecting Cas9 protein and a single guide RNA into fertilized zebrafish eggs, subsequently confirming the presence of a 6- or 9-bp deletion in the ints14 gene. In addition, we examined the two mutant alleles through PCR analysis, T7E1 assay, TA-cloning, and sequencing. For the first time, we used the CRISPR/Cas9 system to create a model in which some sequences of the ints14 gene were removed. This breakthrough opens new avenues for in-depth exploration of the role of ints14 in animal diseases. The mutant strains generated in this study can provide a valuable resource for further investigations into the specific consequences of ints14 gene deletion during zebrafish development. This research establishes a foundation for future studies exploring the molecular mechanisms underlying the functions of ints14, its interactions with other genes or proteins, and its broader implications for biological processes.

• Journal of Embryo Transfer
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• v.29 no.4
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• pp.339-344
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• 2014

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein (Cas9) system can be applied to produce transgenic pigs. Therefore, we applied CRISPR/Cas9 system to generate FoxN1-targeted pig parthenogenetic embryos. Using single guided RNA targeted to pig FoxN1 genes was injected into cytoplasm of in vitro matured oocyte before electrical activation. In results, regardless of the concentrations of vector, the cleavage rate were significantly (p<0.05) decreased ($4ng/{\mu}l$, 51.24%; $8ng/{\mu}l$, 40.88%; and $16ng/{\mu}l$; 45.22%) compared to no injection group (70.44%). The blastocyst formation rates were also decreased in vector injected 3 groups ($4ng/{\mu}l$, 7.96%; $8ng/{\mu}l$, 6.4%; and $16ng/{\mu}l$; 9.04%) compared to no injection group (29.07%). In addition, the blastocyst formation rates between sham injected group (13.51%) and no injection group (29.07%) also showed significant difference (p<0.05). The mutation rates were comparable between groups ($4ng/{\mu}l$, 18.4%; $8ng/{\mu}l$, 12.5%; and $16ng/{\mu}l$; 20.0%). The sequencing analysis showed that blastocysts derived from each group were successfully mutated in FoxN1 loci regardless of the vector concentrations. However, the deletion patterns were higher than the patterns of point mutation and insertion regardless of the vector concentrations. In conclusion, we described that cytoplasmic microinjection of FoxN1-targeted CRISPR/Cas9 vector could efficiently generate transgenic pig parthenogenetic embryos in one-step.

• Genomics & Informatics
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• v.18 no.4
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• pp.46.1-46.4
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• 2020

We developed the BaSDAS (Barcode-Seq Data Analysis System), a GUI-based pooled knockout screening data analysis system, to facilitate the analysis of pooled knockout screen data easily and effectively by researchers with limited bioinformatics skills. The BaSDAS supports the analysis of various pooled screening libraries, including yeast, human, and mouse libraries, and provides many useful statistical and visualization functions with a user-friendly web interface for convenience. We expect that BaSDAS will be a useful tool for the analysis of genome-wide screening data and will support the development of novel drugs based on functional genomics information.

• Journal of Embryo Transfer
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• v.33 no.4
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• pp.245-254
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• 2018

Pigs are considered as optimal donor animal for the successful xenotransplantation. To increase the possibility of clinical application, genetic modification to increase compatibility with human is an important and essential process. Genetic modification technique has been developed and improved to produce genetically modified pigs rapidly. CRISPR/Cas9 system is widely used in various fields including the production of transgenic animals and also can be enable multiple gene modifications. In this study, we developed new gene targeting vector and enrichment system for the rapid and efficient selection of genetically modified cells. We conducted co-transfection with two targeting vectors for simultaneous inactivation of two genes and enrichment of the genetically modified cells using MACS. After this efficient enrichment, genotypic analysis of each colony showed that colonies which have genetic modifications on both genes were confirmed with high efficiency. Somatic cell nuclear transfer was conducted with established donor cells and genetically modified pigs were successfully produced. Genotypic and phenotypic analysis of generated pigs showed identical genotypes with donor cells and no surface expression of ${\alpha}$-Gal and HD antigens. Furthermore, functional analysis using pooled human serum revealed dramatically reduction of human natural antibody (IgG and IgM) binding level and natural antibody-mediated cytotoxicity. In conclusion, the constructed vector and enrichment system using MACS used in this study is efficient and useful to generate genetically modified donor cells with multiple genetic alterations and lead to an efficient production of genetically modified pigs.

• Journal of Plant Biotechnology
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• v.48 no.1
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• pp.34-43
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• 2021

Targeted genome editing using the CRISPR/Cas9 system is a ground-breaking technology that is being widely used to produce plants with useful traits. However, for woody plants, only a few successful attempts have been reported. These successes have used Agrobacterium-mediated transformation, which has been reported to be very efficient at producing genetically modified trees. Nonetheless, there are unresolved problems with plasmid sequences that remain in the plant genome. In this study, we demonstrated a DNA-free genome editing technique in which purified CRISPR/Cas9 ribonucleoproteins (RNPs) are delivered directly to the protoplasts of a hybrid poplar (Populus alba × Populus glandulosa). We designed three single-guide RNAs (sgRNAs) to target the stress-associated protein 1 gene (PagSAP1) in the hybrid poplar. Deep sequencing results showed that pre-assembled RNPs had a more efficient target mutagenesis insertion and deletion (indel) frequency than did non-assembled RNPs. Moreover, the RNP of sgRNA3 had a significantly higher editing efficacy than those of sgRNA1 and sgRNA2. Our results suggest that the CRISPR/Cas9 ribonucleoprotein-mediated transfection approach is useful for the production of transgene-free genome-edited tree plants.

• Animal Bioscience
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• v.35 no.1
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• pp.126-137
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• 2022

Objective: Efficient gene editing technology is critical for successful knock-in in domestic animals. RAD51 recombinase (RAD51) gene plays an important role in strand invasion during homologous recombination (HR) in mammals, and is regulated by checkpoint kinase 1 (CHK1) and CHK2 genes, which are upstream elements of RAD51 recombinase (RAD51). In addition, mismatch repair (MMR) system is inextricably linked to HR-related pathways and regulates HR via heteroduplex rejection. Thus, the aim of this study was to investigate whether clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9)-mediated knock-in efficiency of human lactoferrin (hLF) knock-in vector in the bovine β-casein gene locus can be increased by suppressing DNA MMR-related genes (MSH2, MSH3, MSH6, MLH1, and PMS2) and overexpressing DNA double-strand break (DSB) repair-related genes (RAD51, CHK1, CHK2). Methods: Bovine mammary epithelial (MAC-T) cells were transfected with a knock-in vector, RAD51, CHK1, or CHK2 overexpression vector and CRISPR/sgRNA expression vector to target the bovine β-casein gene locus, followed by treatment of the cells with CdCl₂ for 24 hours. After 3 days of CdCl₂ treatment, the knock-in efficiency was confirmed by polymerase chain reaction (PCR). The mRNA expression levels of DNA MMR-related and DNA DSB repair-related genes were assessed by quantitative real-time PCR (RT-qPCR). Results: Treatment with CdCl₂ decreased the mRNA expression of RAD51 and MMRrelated genes but did not increase the knock-in efficiency in MAC-T cells. Also, the overexpression of DNA DSB repair-related genes in MAC-T cells did not significantly affect the mRNA expression of MMR-related genes and failed to increase the knock-in efficiency. Conclusion: Treatment with CdCl₂ inhibited the mRNA levels of RAD51 and DNA MMR-related genes in MAC-T cells. However, the function of MMR pathway in relation to HR may differ in various cell types or species.

• Proceedings of the Korean Society of Crop Science Conference
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• 2022.10a
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• pp.194-194
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• 2022

Recently, food-induced allergies have emerged as major global concerns. In the past ten years, it has doubled in western nations, and it has also increased in Asia and Africa. In many cases of food allergy, peanut allergy is prevalent, typically permanent, and frequently life-threatening. Therefore, we utilized gene editing techniques on the three major allergen genes in peanuts, Ara h 1, Ara h 2, and Ara h 3. Using gibson assembly and golden gate assembly, we created two vectors, the gRNA-tRNA array CRISPR-Cas9 system and Prime-editing. Using LBA4404 strain and agrobacterium-mediated transformation, the vectors were transferred to two elite Korean peanut lines. After co-cultivation and tissue culture, we extracted the tissue cultured peanut DNA amplified the hygromycin resistance gene and Cas9 gene in the T-DNA region. The integration of the T-DNA region into the host genome was demonstrated by the presence of a specific band in some samples. There have only been a few reported peanut gene editing studies. So, this study will contribute to peanut allergy and gene editing research.

• Proceedings of the Korean Society of Crop Science Conference
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• 2022.10a
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• pp.200-200
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• 2022

Recently, food-induced allergies have emerged as major global concerns. In the past ten years, it has doubled in western nations, and it has also increased in Asia and Africa. In many cases of food allergy, peanut allergy is prevalent, typically permanent, and frequently life-threatening. Therefore, we utilized gene editing techniques on the three major allergen genes in peanuts, Ara h 1, Ara h 2, and Ara h 3. Using gibson assembly and golden gate assembly, we created two vectors, the gRNA-tRNA array CRISPR-Cas9 system and Prime-editing. Using LBA4404 strain and agrobacterium-mediated transformation, the vectors were transferred to two elite Korean peanut lines. After co-cultivation and tissue culture, we extracted the tissue cultured peanut DNA amplified the hygromycin resistance gene and Cas9 gene in the T-DNA region. The integration of the T-DNA region into the host genome was demonstrated by the presence of a specific band in some samples. There have only been a few reported peanut gene editing studies. So, this study will contribute to peanut allergy and gene editing research.