• Journal of Plant Biotechnology
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• 제42권3호
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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.

• Journal of Microbiology and Biotechnology
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• 제26권2호
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• pp.213-225
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• 2016

To reduce attrition in drug development, it is crucial to consider the development and implementation of translational phenotypic assays as well as decipher diverse molecular mechanisms of action for new molecular entities. High-throughput fluorescence and confocal microscopes with advanced analysis software have simplified the simultaneous identification and quantification of various cellular processes through what is now referred to as high-content screening (HCS). HCS permits automated identification of modifiers of accessible and biologically relevant targets and can thus be used to detect gene interactions or identify toxic pathways of drug candidates to improve drug discovery and development processes. In this review, we summarize several HCS-compatible, biochemical, and molecular biology-driven assays, including immunohistochemistry, RNAi, reporter gene assay, CRISPR-Cas9 system, and protein-protein interactions to assess a variety of cellular processes, including proliferation, morphological changes, protein expression, localization, post-translational modifications, and protein-protein interactions. These cell-based assay methods can be applied to not only 2D cell culture but also 3D cell culture systems in a high-throughput manner.

• Korean Chemical Engineering Research
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• 제56권1호
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• pp.79-84
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• 2018

본 연구에서는 기 개발된 액적 접촉충전 기반의 디지털 전기천공 기술을 이용해 미세조류에 단백질을 전달하는 연구를 수행 하였다. Chlamydomonas reinhardtii 중 세포벽이 존재하는 야생종 cc-125에 적용한 결과, 살아 있는 세포의 핵 내부로 형광 단백질 GFP가 10% 이상의 비교적 높은 효율로 전달될 수 있음을 확인하였다. 또한 인가 전기장의 크기 변화에 따른 단백질 전달 효율을 살펴봄으로써 최적의 단백질 전달 효율을 위한 전기천공 전기장 조건을 도출하였다(960 V/cm). 전달 물질의 크기에 따른 영향 분석을 위해 추가로 수행한 핵산 염색 형광 염료 Yo-Pro-1의 전달 특성 분석 결과, 크기에 따른 차이가 존재함에도 최적의 전달 효율을 나타내는 인가 전기장의 세기 조건은 매우 유사한 경향을 보였다. 마지막으로 본 연구 결과의 의미 및 크리스퍼 유전자 가위 기술의 적용 등 향후 활용방안에 대해서 논의하였다.

• Experimental and Molecular Medicine
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• 제50권12호
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• pp.6.1-6.10
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• 2018

Bmal1 is one of the key molecules that controls the mammalian molecular clock. In humans, two isoforms of Bmal1 are generated by alternative RNA splicing. Unlike the extensively studied hBmal1b, the canonical form of Bmal1 in most species, the expression and/or function of another human-specific isoform, hBmal1a, are poorly understood. Due to the lack of the N-terminal nuclear localization signal (NLS), hBMAL1a does not enter the nucleus as hBMAL1b does. However, despite the lack of the NLS, hBMAL1a still dimerizes with either hCLOCK or hBMAL1b and thereby promotes cytoplasmic retention or protein degradation, respectively. Consequently, hBMAL1a interferes with hCLOCK:hBMAL1b-induced transcriptional activation and the circadian oscillation of Period2. Moreover, when the expression of endogenous hBmal1a is aborted by CRISPR/Cas9-mediated knockout, the rhythmic expression of hPer2 and hBmal1b is restored in cultured HeLa cells. Together, these results suggest a role for hBMAL1a as a negative regulator of the mammalian molecular clock.

• Molecules and Cells
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• 제42권11호
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• pp.739-746
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• 2019

Significant knowledge about the pathophysiology of Alzheimer's disease (AD) has been gained in the last century; however, the understanding of its causes of onset remains limited. Late-onset AD is observed in about 95% of patients, and APOE4-encoding apolipoprotein E4 (ApoE4) is strongly associated with these cases. As an apolipoprotein, the function of ApoE in brain cholesterol transport has been extensively studied and widely appreciated. Development of new technologies such as human-induced pluripotent stem cells (hiPSCs) and CRISPR-Cas9 genome editing tools have enabled us to develop human brain model systems in vitro and readily manipulate genomic information. In the context of these advances, recent studies provide strong evidence that abnormal cholesterol metabolism by ApoE4 could be linked to AD-associated pathology. In this review, we discuss novel discoveries in brain cholesterol dysregulation by ApoE4. We further elaborate cell type-specific roles in cholesterol regulation of four major brain cell types, neurons, astrocytes, microglia, and oligodendrocytes, and how its dysregulation can be linked to AD pathology.

• The Plant Pathology Journal
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• 제37권5호
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• pp.494-501
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• 2021

Pseudomonas cichorii secretes effectors that suppress defense mechanisms in host plants. However, the function of these effectors, including avirulence protein E1 (AvrE1), in the pathogenicity of P. cichorii, remains unexplored. In this study, to investigate the function of avrE1 in P. cichorii JBC1 (PcJBC1), we created an avrE1-deficient mutant (JBC1^ΔavrE1) using CRISPR/Cas9. The disease severity caused by JBC1^ΔavrE1 in tomato plants significantly decreased by reducing water soaking during early infection stage, as evidenced by the electrolyte leakage in infected leaves. The disease symptoms caused by JBC1^ΔavrE1 in the cabbage midrib were light-brown spots compared to the dark-colored ones caused by PcJBC1, which indicates the role of AvrE1 in cell lysis. The avrE1-deficient mutant failed to elicit cell death in non-host tobacco plants. Disease severity and cell death caused by JBC1^ΔavrE1 in host and non-host plants were restored through heterologous complementation with avrE1 from Pseudomonas syringae pv. tomato DC3000 (PstDC3000). Overall, our results indicate that avrE1 contributes to cell death during early infection, which consequently increases disease development in host plants. The roles of PcJBC1 AvrE1 in host cells remain to be elucidated.

• Molecules and Cells
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• 제44권7호
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• pp.444-457
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• 2021

Although the mechanism of chronic myeloid leukemia (CML) initiation through BCR/ABL oncogene has been well characterized, CML cell differentiation into erythroid lineage cells remains poorly understood. Using CRISPR-Cas9 screening, we identify Chromobox 8 (CBX8) as a negative regulator of K562 cell differentiation into erythrocytes. CBX8 is degraded via proteasomal pathway during K562 cell differentiation, which activates the expression of erythroid differentiation-related genes that are repressed by CBX8 in the complex of PRC1. During the differentiation process, the serine/threonine-protein kinase PIM1 phosphorylates serine 196 on CBX8, which contributes to CBX8 reduction. When CD235A expression levels are analyzed, the result reveals that the knockdown of PIM1 inhibits K562 cell differentiation. We also identify TRIM28 as another interaction partner of CBX8 by proteomic analysis. Intriguingly, TRIM28 maintains protein stability of CBX8 and TRIM28 loss significantly induces proteasomal degradation of CBX8, resulting in an acceleration of erythroid differentiation. Here, we demonstrate the involvement of the CBX8-TRIM28 axis during CML cell differentiation, suggesting that CBX8 and TRIM28 are promising novel targets for CML research.

• Molecules and Cells
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• 제42권7호
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• pp.546-556
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• 2019

Protein phosphatase 4 (PP4) is a crucial protein complex that plays an important role in DNA damage response (DDR), including DNA repair, cell cycle arrest and apoptosis. Despite the significance of PP4, the mechanism by which PP4 is regulated remains to be elucidated. Here, we identified a novel PP4 inhibitor, protein phosphatase 4 inhibitory protein (PP4IP) and elucidated its cellular functions. PP4IP-knockout cells were generated using the CRISPR/Cas9 system, and the phosphorylation status of PP4 substrates (H2AX, KAP1, and RPA2) was analyzed. Then we investigated that how PP4IP affects the cellular functions of PP4 by immunoprecipitation, immunofluorescence, and DNA double-strand break (DSB) repair assays. PP4IP interacts with PP4 complex, which is affected by DNA damage and cell cycle progression and decreases the dephosphorylational activity of PP4. Both overexpression and depletion of PP4IP impairs DSB repairs and sensitizes cells to genotoxic stress, suggesting timely inhibition of PP4 to be indispensable for cells in responding to DNA damage. Our results identify a novel inhibitor of PP4 that inhibits PP4-mediated cellular functions and establish the physiological importance of this regulation. In addition, PP4IP might be developed as potential therapeutic reagents for targeting tumors particularly with high level of PP4C expression.

• Journal of Microbiology and Biotechnology
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• 제29권5호
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• pp.667-686
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• 2019

Streptomyces are attractive microbial cell factories that have industrial capability to produce a wide array of bioactive secondary metabolites. However, the genetic potential of the Streptomyces species has not been fully utilized because most of their secondary metabolite biosynthetic gene clusters (SM-BGCs) are silent under laboratory culture conditions. In an effort to activate SM-BGCs encoded in Streptomyces genomes, synthetic biology has emerged as a robust strategy to understand, design, and engineer the biosynthetic capability of Streptomyces secondary metabolites. In this regard, diverse synthetic biology tools have been developed for Streptomyces species with technical advances in DNA synthesis, sequencing, and editing. Here, we review recent progress in the development of synthetic biology tools for the production of novel secondary metabolites in Streptomyces, including genomic elements and genome engineering tools for Streptomyces, the heterologous gene expression strategy of designed biosynthetic gene clusters in the Streptomyces chassis strain, and future directions to expand diversity of novel secondary metabolites.

• The Plant Pathology Journal
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• 제37권4호
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• pp.389-395
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• 2021

Soil is the major source of plant-associated microbes. Several fungal and bacterial species live within plant tissues. Actinomycetes are well known for producing a variety of antibiotics, and they contribute to improving plant health. In our previous report, Streptomyces globisporus SP6C4 colonized plant tissues and was able to move to other tissues from the initially colonized ones. This strain has excellent antifungal and antibacterial activities and provides a suppressive effect upon various plant diseases. Here, we report the genome-wide analysis of antibiotic producing genes in S. globisporus SP6C4. A total of 15 secondary metabolite biosynthetic gene clusters were predicted using antiSMASH. We used the CRISPR/Cas9 mutagenesis system, and each biosynthetic gene was predicted via protein basic local alignment search tool (BLAST) and rapid annotation using subsystems technology (RAST) server. Three gene clusters were shown to exhibit antifungal or antibacterial activity, viz. cluster 16 (lasso peptide), cluster 17 (thiopeptide-lantipeptide), and cluster 20 (lantipeptide). The results of the current study showed that SP6C4 has a variety of antimicrobial activities, and this strain is beneficial in agriculture.