• Korean Journal of Agricultural Science
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• v.46 no.4
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• pp.885-895
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• 2019

Plant biotechnologists have long dreamed of technologies to manipulate genes in plants at will. This dream has come true partly through the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology, which now has been used to edit genes in several important crops. However, there are many restrictions in editing a gene precisely using the CRISPR/Cas9 technology because CRISPR/Cas9 may cause deletions or additions in some regions of the target gene. Several other technologies have been developed for gene targeting and precision editing. Among these, base editors might be the most practically and efficiently used compared to others. Base editors are tools which are able to cause a transition from cytosine into thymine, or from adenine into guanine very precisely on specific sequences. Cytosine base editors basically consist of nCas9, cytosine deaminase, and uracil DNA glycosylase inhibitor (UGI). Adenine base editors consist of nCas9 and adenine deaminase. These were first developed for human cells and have since also been applied successfully to crops. Base editors have been successfully applied for productivity improvement, fortification and herbicide resistance of crops. Thus, base editor technologies start to open a new era for precision gene editing or breeding in crops and might result in revolutionary changes in crop breeding and biotechnology.

• Korean Journal of Environmental Agriculture
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• v.40 no.3
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• pp.179-185
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• 2021

BACKGROUND: Tomato genome editing using CRISPR-Cas9 is being actively conducted in recent days, and lots of plant researches have been aiming to develop high valued crops by editing target genes without inserting foreign genes. Many researchers have been involved in the manipulation of the crop ripening process because fruit ripening is an important fruit phenotype for increasing fruit shelf life, taste, and texture of crops. This paper intends to evaluate target sgRNA to edit the two ripening-related genes encoding pectate lyase (PL) and phytoene synthase (Psy) with the CRISPR-Cas9 system. METHODS AND RESULTS: The CRISPR-Cas9 expression vector was cloned to target the PL (Solyc03g111690), Psy1 (Solyc03g031860), and Psy2 (Solyc02g081330) genes, which are the ripening genes of tomatoes. Tomatoes injected with Agrobacterium containing the CRISPR-Cas9 expression vector were further cultured for 5 days and used to check gene editing efficiency. As a result of the target gene sequence analysis by the next generation sequencing method, gene editing efficiency was calculated, and the efficient target location was selected for the PL and Psy genes. CONCLUSION: Therefore, this study was aimed to establish target sgRNA data that could have higher efficiency of the CRISPR-Cas9 system to obtain the delayed ripening phenotype of tomato. The developed method and sgRNA information is expected to be utilized in the development of various crops to manage its ripening processes.

• 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.

• Journal of Ginseng Research
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• v.46 no.4
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• pp.505-514
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• 2022

Background: The roots of Panax ginseng contain two types of tetracyclic triterpenoid saponins, namely, protopanaxadiol (PPD)-type saponins and protopanaxatiol (PPT)-type saponins. In P. ginseng, the protopanaxadiol 6-hydroxylase (PPT synthase) enzyme catalyses protopanaxatriol (PPT) production from protopanaxadiol (PPD). In this study, we constructed homozygous mutant lines of ginseng by CRISPR/Cas9-mediated mutagenesis of the PPT synthase gene and obtained the mutant ginseng root lines having complete depletion of the PPT-type ginsenosides. Methods: Two sgRNAs (single guide RNAs) were designed for target mutations in the exon sequences of the two PPT synthase genes (both PPTa and PPTg sequences) with the CRISPR/Cas9 system. Transgenic ginseng roots were generated through Agrobacterium-mediated transformation. The mutant lines were screened by ginsenoside analysis and DNA sequencing. Result: Ginsenoside analysis revealed the complete depletion of PPT-type ginsenosides in three putative mutant lines (Cr4, Cr7, and Cr14). The reduction of PPT-type ginsenosides in mutant lines led to increased accumulation of PPD-type ginsenosides. The gene editing in the selected mutant lines was confirmed by targeted deep sequencing. Conclusion: We have established the genome editing protocol by CRISPR/Cas9 system in P. ginseng and demonstrated the mutated roots producing only PPD-type ginsenosides by depleting PPT-type ginsenosides. Because the pharmacological activity of PPD-group ginsenosides is significantly different from that of PPT-group ginsenosides, the new type of ginseng mutant producing only PPD-group ginsenosides may have new pharmacological characteristics compared to wild-type ginseng. This is the first report to generate target-induced mutations for the modification of saponin biosynthesis in Panax species using CRISPR-Cas9 system.

• Proceedings of the Korean Society of Crop Science Conference
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• 2022.10a
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• pp.285-285
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• 2022

Recent unpredictable climate change is a major cause of rice yield loss. In particular, methane is a key factor in global warming. Therefore rice breeders are trying to breed the reducing-methane gas emission rice using the crossbreeding method. However, the traditional crossbreeding method takes 8 to 10 years to breed a cultivar, and the anther culture method developed to shorten the breeding cycle also takes 6 to 7 years. On the other hand, CRISPR/Cas9 accurately edits the target trait and can rapidly breed rice cultivars by editing the target trait as a homozygous in 2-3 years. In addition, exogenous genetic elements such as Cas9 can be isolated from the G₁ generation. Therefore, the flowering time was regulated by applying CRISPR/Cas9 technology, and OsCKq1 genome-editing (OsCKq1-G) rice with early flowered and high yield was bred in the field. Genome-editing of OsCKq1 applied CRISPR/Cas9 technology up-regulates the expression of the flowering promotion gene Ehd1 under long-day conditions induces early flowering and increases the yield by increasing the 1,000-grain weight. And as the generations advanced, each agricultural trait indicated a low coefficient of variation. As a result, indicated that OsCKq1 plays an important role in regulating the flowering time and is related to the trait determining yield. Therefore, OsCKq1-G can suggest a breeding strategy for the Net-Zero national policy for reducing-methane gas emission rice by shortening the breeding cycle with the early flowered, and high-yield rice. CRISPR/Cas9 technology is a rapid and accurate breeding technology for breeding rice cultivars with important characteristics.

• Proceedings of the Korean Society of Crop Science Conference
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• 2022.10a
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• pp.288-288
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• 2022

Rice is an important staple in the world. And drought is one of the important constraints that negatively affect yield loss and grain quality of rice. CRISPR/Cas9 is a new breeding strategy that can improve the characteristics of rice quickly and accurately. CRISPR/Cas9 is a novel approach that can reliably harvest rice yields in response to a rapidly changing climate. In addition, there is no externally inserted DNA left in genome-editing rice, and it is receiving attention as being able to take responsibility for future food because its characteristics are continuously improved. In the future, high levels of drought resistant in water-constrained environments will be required, which will reduce yield loss. OsSAP was genome-editing with CRISPR/Cas9 in rice. A different line number was assigned to each panicle, and the generation advanced by applying the ear-to-row method. Genome-editing rice has improved drought resistance in drought conditions. Also, in genome-editing rice, the target sequence was homozygous in the 0 generation, and the coefficient of variation of heading date, number of tiller, and 1,000-grain weight was very small in 2 generation. In the era of rapidly changing climate change, CRISPR/Cas9 presents a new breeding strategy that can rapidly and accurately improve agronomic traits of major food crops as well as rice. CRISPR/Cas9 is applied together with traditional breeding to develop into a new breeding strategy, it is suggested that food can be obtained stably in response to climate change.

• Journal of Life Science
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• v.30 no.3
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• pp.313-319
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• 2020

Recently, cattle epidemic diseases are caused by a pathogen such as a virus or bacterium. Such diseases can spread through various pathways, such as feed intake, respiration, and contact between livestock. Diagnosis based on the ELISA (Enzyme-linked immunosorbent assay) and PCR (Polymerase chain reaction) methods has limitations because these traditional diagnostic methods are time consuming assays that require multiple steps and dedicated equipment. In this review, we propose the use of the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) Cas system based on DNA and RNA levels for early point-of-care diagnosis in cattle. In the CRISPR/Cas system, Cas effectors are classified into two classes and six subtypes. The Cas effectors included in class 2 are typically Cas9 in type II, Cas12 in type V (Cas12a and Cas12b) and Cas13 in type VI (Cas13a and Cas13b). The CRISPR/Cas system uses reporter molecules that are attached to the ssDNA strands. When the Cas enzyme cuts the ssDNA, these reporters either fluoresce or change color, indicating the presence of a specific disease marker. There are several steps in the development of a CRISPR/Cas system. The first is to select the Cas enzyme depending on DNA or RNA from pathogens (viruses or bacteria). Based on that, the next step is to integrate the optimal amplification, transducing method, and signal reporter. The CRISPR/Cas system is a powerful diagnostic tool using a gene-editing method, which is faster, better, and cheaper than traditional methods. This system could be used for early diagnosis of epidemic cattle diseases and help to control their spread.

• Korean Journal of Environment and Ecology
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• v.35 no.5
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• pp.503-524
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• 2021

A largemouth bass (Micropterus salmoides) is an ecosystem disturbance fish species at the highest rank in the aquatic ecosystem, causing a serious imbalance in freshwater ecosystems. Although various attempts have been made to eradicate and control largemouth bass, no effective measures were found. Therefore, it is necessary to find an approach to maximize the effective population reduction based on the unique characteristics of largemouth bass. This study used the transcriptome analysis to derive 182,887 unigene contigs and select 12 types of final target sequences for applying the CRISPR/Cas9 system in the genes of IZUMO1 and Zona pellucida sperm-binding protein, which are proteins involved in sperm-egg recognition. After synthesizing 12 types of sgRNA capable of recognizing each target sequence, 12 types of Cas9-sgRNA ribonucleoprotein (RNP) complexes to be used in subsequent studies were prepared. This study searched the protein-coding gene of sperm-egg through the Next Generation Sequencing (NGS) and edited genes through the CRISPR/Cas9 system to induce infertile individuals that produced reproductive cells but could not form fertilized eggs. Through such a series of processes, it successfully established a composition development process for largemouth bass. It is judged that this study contributed to securing the valuable basic data for follow-up studies to verify its effect for the management of ecological disturbances without affecting the habitat of other endemic species in the same water system with the largemouth bass.

• BMB Reports
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• v.53 no.7
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• pp.341-348
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• 2020

The targeted nuclease clustered, regularly interspaced short palindromic repeats/CRISPR-associated proteins (CRISPR/Cas) system has recently emerged as a prominent gene manipulation method. Because of its ease in programming targeted DNA/protein binding through RNA in a vast range of organisms, this prokaryotic defense system is a versatile tool with many applications in the research field as well as high potential in agricultural and clinical improvements. This review will present a brief history that led to its discovery and adaptation. We also present some of its restrictions, and modifications that have been performed to overcome such restrictions, focusing specifically on the most common CRISPR/Cas9 mediated non-homologous end joint repair.

• Environmental Mutagens and Carcinogens
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• v.24 no.2
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• pp.99-107
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• 2004

The validation of many synthetic chemicals that may pose a genetic hazard in our environment is of great concern at present. Since these substances are not limited to the original products, and enter the environment, they have become widespread environmental pollutants, thus leading to a variety of chemicals that possibly threaten the public health. In this respect, the regulation and evaluation of the chemical hazard playa very important role to environment and human health. The clastogenicity of 11 synthetic chemicals was evaluated in Chinese hamster lung (CHL) fibroblast in vitro. Benzoyl chloride (CAS No. 98-88-4) induced chromosomal aberrations with statistical significance at the concentration of 31-123 $\mug/ml$ and 43 $\mug/ml$ in the absence and presence of S-9 metabolic activation system, respectively. 2-Propyn-l-o1 (CAS No. 107-19-7) and 2-Phenoxy ethanol (CAS No. 122-99-6) revealed clastogenicity only at the highest concentration in the presence of S-9 mixture. However, 1-naphthol (CAS No. 90-15-3) which is one of the most cytotoxic chemical among 11 chemicals tested revealed no clastogenicity both in the presence and absence of S-9 metabolic activation system. From the results of chromosomal aberration assay with 11 synthetic chemicals in CHL fibroblast in vitro, Benzoyl chloride (CAS No. 98-88-4), 2-Propyn-l-01 (CAS No. 107-19-7) and 2-Phenoxy ethanol (CAS No. 122-99-6) revealed positive clastogenic results in this study.