• Title/Summary/Keyword: CRISPR-Cas9

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Drug evaluation based on phosphomimetic PDHA1 reveals the complexity of activity-related cell death in A549 non-small cell lung cancer cells

  • Jin, Ling;Cho, Minkyoung;Kim, Bo-Sung;Han, Jung Ho;Park, Sungmi;Lee, In-Kyu;Ryu, Dongryeol;Kim, Jae Ho;Bae, Sung-Jin;Ha, Ki-Tae
    • BMB Reports
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    • v.54 no.11
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    • pp.563-568
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    • 2021
  • Cancer cells predominantly generate energy via glycolysis, even in the presence of oxygen, to support abnormal cell proliferation. Suppression of PDHA1 by PDK1 prevents the conversion of cytoplasmic pyruvate into Acetyl-CoA. Several PDK inhibitors have been identified, but their clinical applications have not been successful for unclear reasons. In this study, endogenous PDHA1 in A549 cells was silenced by the CRISPR/Cas9 system, and PDHA1WT and PDHA13SD were transduced. Since PDHA13SD cannot be phosphorylated by PDKs, it was used to evaluate the specific activity of PDK inhibitors. This study highlights that PDHA1WT and PDHA13SD A549 cells can be used as a cell-based PDK inhibitor-distinction system to examine the relationship between PDH activity and cell death by established PDK inhibitors. Leelamine, huzhangoside A and otobaphenol induced PDH activity-dependent apoptosis, whereas AZD7545, VER-246608 and DCA effectively enhanced PDHA1 activity but little toxic to cancer cells. Furthermore, the activity of phosphomimetic PDHA1 revealed the complexity of its regulation, which requires further in-depth investigation.

Development of a Novel ATP Bioluminescence Assay Based on Engineered Probiotic Saccharomyces boulardii Expressing Firefly Luciferase

  • Ji Sun Park;Young-Woo Kim;Hyungdong Kim;Sun-Ki Kim;Kyeongsoon Park
    • Journal of Microbiology and Biotechnology
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    • v.33 no.11
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    • pp.1506-1512
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    • 2023
  • Quantitative analysis of adenosine triphosphate (ATP) has been widely used as a diagnostic tool in the food and medical industries. Particularly, the pathogenesis of a few diseases including inflammatory bowel disease (IBD) is closely related to high ATP concentrations. A bioluminescent D-luciferin/luciferase system, which includes a luciferase (FLuc) from the firefly Photinus pyralis as a key component, is the most commonly used method for the detection and quantification of ATP. Here, instead of isolating FLuc produced in recombinant Escherichia coli, we aimed to develop a whole-cell biocatalyst system that does not require extraction and purification of FLuc. To this end, the gene coding for FLuc was introduced into the genome of probiotic Saccharomyces boulardii using the CRISPR/Cas9-based genome editing system. The linear relationship (r2 = 0.9561) between ATP levels and bioluminescence generated from the engineered S. boulardii expressing FLuc was observed in vitro. To explore the feasibility of using the engineered S. boulardii expressing FLuc as a whole-cell biosensor to detect inflammation biomarker (i.e., ATP) in the gut, a colitis mouse model was established using dextran sodium sulfate as a colitogenic compound. Our findings demonstrated that the whole-cell biosensor can detect elevated ATP levels during gut inflammation in mice. Therefore, the simple and powerful method developed herein could be applied for non-invasive IBD diagnosis.

Differentiation and Characterization of Cystic Fibrosis Transmembrane Conductance Regulator Knockout Human Pluripotent Stem Cells into Salivary Gland Epithelial Progenitors

  • Shuang Yan;Yifei Zhang;Siqi Zhang;Shicheng Wei
    • International Journal of Stem Cells
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    • v.16 no.4
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    • pp.394-405
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    • 2023
  • The differentiation of pluripotent stem cells has been used to study disease mechanisms and development. We previously described a method for differentiating human pluripotent stem cells (hPSCs) into salivary gland epithelial progenitors (SGEPs). Here, cystic fibrosis transmembrane conductance regulator (CFTR) knockout hPSCs were differentiated into SGEPs derived from CFTR knockout hESCs (CF-SGEPs) using the same protocol to investigate whether the hPSC-derived SGEPs can model the characteristics of CF. CF-a disease that affects salivary gland (SG) function-is caused by mutations of the CFTR gene. Firstly, we successfully generated CFTR knockout hPSCs with reduced CFTR protein expression using the CRISPR-Cas9 system. After 16 days of differentiation, the protein expression of CFTR decreased in SGEPs derived from CFTR knockout hESCs (CF-SGEPs). RNA-Seq revealed that multiple genes modulating SG development and function were down-regulated, and positive regulators of inflammation were up-regulated in CF-SGEPs, correlating with the salivary phenotype of CF patients. These results demonstrated that CFTR suppression disrupted the differentiation of hPSC-derived SGEPs, which modeled the SG development of CF patients. In summary, this study not only proved that the hPSC-derived SGEPs could serve as manipulable and readily accessible cell models for the study of SG developmental diseases but also opened up new avenues for the study of the CF mechanism.

Collagen-Induced Arthritis Analysis in Rhbdf2 Knockout Mouse

  • Lee, Min-Young;Kang, Ju-Seong;Go, Ryeo-Eun;Byun, Yong-Sub;Wi, Young Jin;Hwang, Kyung-A;Choi, Jae-Hoon;Kim, Hyoung-Chin;Choi, Kyung-Chul;Nam, Ki-Hoan
    • Biomolecules & Therapeutics
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    • v.26 no.3
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    • pp.298-305
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    • 2018
  • Rhomboid family member 2 gene (Rhbdf2) is an inactive homologue lacking essential catalytic residues of rhomboid intramembrane serine proteases. The protein is necessary for maturation of tumor necrosis factor-alpha ($TNF-{\alpha}$) converting enzyme, which is the molecule responsible for the release of $TNF-{\alpha}$. In this study, Rhbdf2 knockout (KO) mice were produced by CRISPR/CAS9. To see the effects of the failure of $TNF-{\alpha}$ release induced by Rhbdf2 gene KO, collagen-induced arthritis (CIA), which is the representative $TNF-{\alpha}$ related disease, was induced in the Rhbdf2 mutant mouse using chicken collagen type II. The severity of the CIA was measured by traditional clinical scores and histopathological analysis of hind limb joints. A rota-rod test and grip strength test were employed to evaluate the severity of CIA based on losses of physical functions. The results indicated that Rhbdf2 mutant mice showed clear alleviation of the clinical severity of CIA as demonstrated by the significantly lower severity indexes. Moreover, a grip strength test was shown to be useful for the evaluation of physical functional losses by CIA. Overall, the results showed that the Rhbdf2 gene has a significant effect on the induction of CIA, which is related to $TNF-{\alpha}$.

Current status on the modification of the scope for GMO regulation on the gene edited plants with no remnants of inserted foreign DNA fragments (외래 DNA단편이 잔존하지 않는 유전자교정식물에 대한 GMO규제 범위의 제외에 관한 국제 동향)

  • Lee, Shin-Woo
    • Journal of Plant Biotechnology
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    • v.46 no.3
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    • pp.137-142
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    • 2019
  • Gene edited crops can be classified as SDN-1, SDN-2 and SDN-3 group depending on their mutation's range and the usage of donor DNA. The SDN-1 and SDN-2 crops, in particular, could be developed as 100% transgene-free, which do not contain any DNA fragment of the vector or guide RNA used for gene editing such as CRISPR Cas9 system. Therefore, there are no scientific methods available for the detection of these crops and differentiation with the one produced by conventional cross breeding techniques. Additionally, it would be impossible to properly implement the existing GMO regulation law, in particular, the national legislation for "GMO labelling". In this regard, Australia has announced that SDN-1 crops will not be subjected to the existing GMO regulation. Furthermore, Argentina and Brazil have established a new policy that GE crops with no transgene (100% transgene-free crops) should be exempted from the scope of the GMO. In addition, Japan has also announced that "an organism that has no remnants of inserted nucleic acid processed extracellularly is not subjected to the Cartagena Act". It means that SDN-2 crops can also be exempted from the scope of GMO. In this trend, in South Korea, I suggested that gene edited crops with no remnants of inserted foreign DNA fragments should be excluded from the existing GMO regulation. Thus, I expect that diverse elite crop lines should be developed by using advanced gene editing technologies

Global Transcriptome-Wide Association Studies (TWAS) Reveal a Gene Regulation Network of Eating and Cooking Quality Traits in Rice

  • Weiguo Zhao;Qiang He;Kyu-Won Kim;Feifei Xu;Thant Zin Maung;Aueangporn Somsri;Min-Young Yoon;Sang-Beom Lee;Seung-Hyun Kim;Joohyun Lee;Soon-Wook Kwon;Gang-Seob Lee;Bhagwat Nawade;Sang-Ho Chu;Wondo Lee;Yoo-Hyun Cho;Chang-Yong Lee;Ill-Min Chung;Jong-Seong Jeon;Yong-Jin Park
    • Proceedings of the Korean Society of Crop Science Conference
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    • 2022.10a
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    • pp.207-207
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    • 2022
  • Eating and cooking quality (ECQ) is one of the most complex quantitative traits in rice. The understanding of genetic regulation of transcript expression levels attributing to phenotypic variation in ECQ traits is limited. We integrated whole-genome resequencing, transcriptome, and phenotypic variation data from 84 Japonica accessions to build a transcriptome-wide association study (TWAS) based regulatory network. All ECQ traits showed a large phenotypic variation and significant phenotypic correlations among the traits. TWAS analysis identified a total of 285 transcripts significantly associated with six ECQ traits. Genome-wide mapping of ECQ-associated transcripts revealed 66,905 quantitative expression traits (eQTLs), including 21,747 local eQTLs, and 45,158 trans-eQTLs, regulating the expression of 43 genes. The starch synthesis-related genes (SSRGs), starch synthase IV-1 (SSIV-1), starch branching enzyme 1 (SBE1), granule-bound starch synthase 2 (GBSS2), and ADP-glucose pyrophosphorylase small subunit 2a (OsAGPS2a) were found to have eQTLs regulating the expression of ECQ associated transcripts. Further, in co-expression analysis, 130 genes produced at least one network with 22 master regulators. In addition, we developed CRISPR/Cas9-edited glbl mutant lines that confirmed the role of alpha-globulin (glbl) in starch synthesis to validate the co-expression analysis. This study provided novel insights into the genetic regulation of ECQ traits, and transcripts associated with these traits were discovered that could be used in further rice breeding.

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