• 한국환경농학회지
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• 제40권3호
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• pp.186-193
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• 2021

BACKGROUND: Before generating transgenic plant using the CRISPR-Cas9 system, the efficiency test of sgRNAs is recommended to reduce the time and effort for plant transformation and regeneration process. The efficiency of the sgRNA can be measured through the transient expression of sgRNA and Cas9 gene in tomato cotyledon; however, we found that the calculated efficiency showed a large variation. It is necessary to increase the precision of the experiment to obtain reliable sgRNA efficiency data from transient expression. METHODS AND RESULTS: The cotyledon of 11^th, 15^th, 19^th, and 23^rd-day-old tomato (Solanum lycopersicum cv. Micro-Tom) were used for expressing CRISPR-Cas9 transiently. The agrobacterium harboring sgRNA for targeting ALS2 gene of tomato was injected through the stomata of leaf adaxial side and the genomic DNA was extracted in 5 days after injection. The target gene edition was identified by amplifying DNA fragment of target region and analyzing with Illumina sequencing method. The target gene editing efficiency was calculated by counting base deletion and insertion events from total target sequence read. CONCLUSION: The CRISPR-Cas9 editing efficiency varied with tomato cotyledon age. The highest efficiency was observed at the 19-day-old cotyledons. Both the median and mean were the highest at this stage and the sample variability was also minimized. We found that the transgene of CRISPR-Cas9 system was strongly correlated with plant leaf development and suggested the optimum cotyledon leaf age for Agrobacterium-mediated transfection in tomato.

• 한국환경농학회지
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• 제40권3호
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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.

• Molecules and Cells
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• 제41권10호
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• pp.917-922
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• 2018

The CRISPR-Cas system is a well-established RNA-guided DNA editing technique widely used to modify genomic DNA sequences. I used the CRISPR-Cas9 system to change the second and third nucleotides of the triplet $T{\underline{CT}}$ of human TNSFSF9 in HepG2 cells to $T{\underline{AG}}$ to create an amber stop codon. The $T{\underline{CT}}$ triplet is the codon for Ser at the $172^{nd}$ position of TNSFSF9. The two substituted nucleotides, AG, were confirmed by DNA sequencing of the PCR product followed by PCR amplification of the genomic TNFSF9 gene. Interestingly, sequencing of the cDNA of transcripts of the edited TNFSF9 gene revealed that the $T{\underline{AG}}$ had been re-edited to the wild type triplet $T{\underline{CT}}$, and 1 or 2 bases just before the triplet had been deleted. These observations indicate that CRISPR-Cas9-mediated editing of bases in target genomic DNA can be followed by spontaneous re-editing (correcting) of the bases during transcription.

• BMB Reports
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• 제54권1호
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• pp.59-69
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• 2021

The ability to read, write, and edit genomic information in living organisms can have a profound impact on research, health, economic, and environmental issues. The CRISPR/Cas system, recently discovered as an adaptive immune system in prokaryotes, has revolutionized the ease and throughput of genome editing in mammalian cells and has proved itself indispensable to the engineering of immune cells and identification of novel immune mechanisms. In this review, we summarize the CRISPR/Cas9 system and the history of its discovery and optimization. We then focus on engineering T cells and other types of immune cells, with emphasis on therapeutic applications. Last, we describe the different modifications of Cas9 and their recent applications in the genome-wide screening of immune cells.

• BMB Reports
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• 제53권7호
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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.

• 한국작물학회:학술대회논문집
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• 한국작물학회 2019년도 춘계학술대회
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• pp.9-9
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• 2019

• 생명과학회지
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• 제30권3호
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• pp.313-319
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• 2020

최근, 국내에서 발생하는 대가축의 질병은 바이러스 혹은 세균 등과 같은 병원체가 사료 섭취, 가축 간의 신체접촉, 호흡 등 다양한 경로를 통해 전파되어 발병되는 전염성 질병이다. 전염성 질병은 가축의 건강을 위협하고 생산성을 감소시키기 때문에 현장에서 조기 진단하여 개체 격리와 같은 통제 관리가 필수적이다. 기존 사용되고 있는 진단 키트들은 현장에서 사용하기에 용이하지 않으며 극소량의 감도에서 진단이 제한적인 단점을 가지고 있다. 그러므로, 현장에서 극소량의 감도와 진단의 편이성을 고려하여 DNA와 RNA 수준에서 진단할 수 있는 CRISPR/Cas 시스템은 최적의 시스템이라 할 수 있다. 본 연구논문에서는 대가축의 전염성 질병들을 현장에서 조기 진단함에 있어 CRISPR/Cas 시스템의 활용전략에 대해 소개하고자 한다. 최근 발견된 CRISPR/Cas 효소들은 2개의 클래스와 6가지 하위유형으로 분류되었다. 이 중에서 클래스 2에 포함되는 Cas 효소들은 대표적으로 제 2형에 Cas9, 제 5형에 Cas12a와 Cas12b, 제 6형에 Cas13a와 Cas13b가 있다. 현재까지 개발된 CRISPR/Cas 시스템들은 간단한 시각 신호를 통해 표적에 대한 정량 및 다중 감지가 가능하고 특히, 극소량 수준의 초고감도에서도 표적만을 진단할 수 있으며 단시간 이내에 진단 결과를 얻을 수 있다. 하지만 초고감도 DNA 혹은 RNA를 진단하기 위해 최적의 신호 증폭 방법과 결합되어야 하고 표적 DNA 혹은 RNA를 진단에 적합하도록 DNA를 RNA로, RNA를 DNA로 전변해야 하는 단점이 있다. 따라서, 현장에서 대가축의 전염성 질병을 조기에 진단할 수 있는 CRISPR/Cas 바이오센서를 개발하는데 있어 가축의 전염 매개체로부터 추출되는 병원체 유형(DNA 혹은 RNA)을 고려하여 최적의 Cas 효소를 선정하여야 하고 이에 따른 적절한 신호 증폭 방법이 결합되어야 한다. 따라서, CRISPR/Cas 시스템은 유전자 편집 방법을 사용하는 빠르고 효율적인 진단 도구이며 이 시스템은 소의 전염병을 조기에 진단하고 감염 확산방지에 도움될 수 있을 것으로 판단 되어진다.

• Molecules and Cells
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• 제46권1호
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• pp.4-9
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• 2023

The advent of the CRISPR-Cas genome editing platform has greatly enhanced the capabilities of researchers in many areas of biology. Its use has also been turned to the development of therapies for genetic diseases and to the enhancement of cell therapies. This review describes some recent advances in these areas.

• Journal of Microbiology and Biotechnology
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• 제31권7호
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• pp.903-911
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• 2021

Previous studies have modified microbial genomes by introducing gene cassettes containing selectable markers and homologous DNA fragments. However, this requires several steps including homologous recombination and excision of unnecessary DNA regions, such as selectable markers from the modified genome. Further, genomic manipulation often leaves scars and traces that interfere with downstream iterative genome engineering. A decade ago, the CRISPR/Cas system (also known as the bacterial adaptive immune system) revolutionized genome editing technology. Among the various CRISPR nucleases of numerous bacteria and archaea, the Cas9 and Cas12a (Cpf1) systems have been largely adopted for genome editing in all living organisms due to their simplicity, as they consist of a single polypeptide nuclease with a target-recognizing RNA. However, accurate and fine-tuned genome editing remains challenging due to mismatch tolerance and protospacer adjacent motif (PAM)-dependent target recognition. Therefore, this review describes how to overcome the aforementioned hurdles, which especially affect genome editing in higher organisms. Additionally, the biological significance of CRISPR-mediated microbial genome editing is discussed, and future research and development directions are also proposed.

• 대한의생명과학회지
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• 제25권4호
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• pp.372-380
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• 2019

Mice with specific modified genes are useful means of studying development and disease. The CRISPR/Cas9 system is a very powerful and effective tool for generating genetically modified mice in a simple and fast manner. To generate human IgG light kappa constant knock-in mice, we tested by microinjection of a mixture of Cas9 protein, single-guide RNA and target homologous recombinant donor DNA into zygotes. We found that the injection of 10 ng/μL of Cas9 protein and crRNA/tracrRNA, rather than single guide RNA, induced the production of knock-in mice more effectively. Thus, our study provides valuable information that will help to improve the production of knock-in mice and contribute the successful generation of humanized Ab-producing mice in Korea.