• BMB Reports
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• v.56 no.2
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• pp.102-107
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• 2023

Genome editing using CRISPR-associated technology is widely used to modify the genomes rapidly and efficiently on specific DNA double-strand breaks (DSBs) induced by Cas9 endonuclease. However, despite swift advance in Cas9 engineering, structural basis of Cas9-recognition and cleavage complex remains unclear. Proper assembly of this complex correlates to effective Cas9 activity, leading to high efficacy of genome editing events. Here, we develop a CRISPR/Cas9-RAD51 plasmid constitutively expressing RAD51, which can bind to single-stranded DNA for DSB repair. We show that the efficiency of CRISPR-mediated genome editing can be significantly improved by expressing RAD51, responsible for DSB repair via homologous recombination (HR), in both gene knock-out and knock-in processes. In cells with CRISPR/Cas9-RAD51 plasmid, expression of the target genes (cohesin SMC3 and GAPDH) was reduced by more than 1.9-fold compared to the CRISPR/Cas9 plasmid for knock-out of genes. Furthermore, CRISPR/Cas9-RAD51 enhanced the knock-in efficiency of DsRed donor DNA. Thus, the CRISPR/Cas9-RAD51 system is useful for applications requiring precise and efficient genome edits not accessible to HR-deficient cell genome editing and for developing CRISPR/Cas9-mediated knockout technology.

• Journal of Sericultural and Entomological Science
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• v.40 no.1
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• pp.52-59
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• 1998

For the construction of plasmid and bmNPV sarrying the FRT recognition site for the FLP recombinases, we synthesized the wild type FRT dligonucleotides. The target FRT sequences consist of three 13bp repeated DNA sequences; two repeats in a direct orientation and one inverted relative to the other two. In addition, there is an 8bp spacer region between the repeats which determune the orientation of the FRT recombination site. In order to place the FRT site both in target BmNPV genome and the transfer vector, we constructed a plasmid, FRT site both in the target BmNPv genome and the transfer vector, we constructed a plasmid, pFRT$\beta$-gal, carrying the FRT sites within the cloning sites of pSV vector and a recombinant BmNPV, vFRTPH, carrying the FRT sites at a downstream of polyhedrin promotor, respectively. In order to test the functionality of the FLP/FRT site-specific recombination system, vFRTPH, pFRT$\beta$-gal and pHsFLP DNA were co-transfected into BmN-4 cells. The resulting recombinant virus was designated a vFRT$\beta$2-gal. From construction analysis of the vFRT$\beta$2-gal with PCR technique it was concluded that the entire pFRT$\beta$-gal plasmid with $\beta$-galactosidase gene and origines of replication flanked by two functional hybrid FRT sequences. The efficiency of recombination was 8.7%, which was higher than that(2.2%) of recombination between a conventional transfer vector and the wild type BmNPV.

• Journal of Microbiology and Biotechnology
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• v.27 no.2
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• pp.335-341
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• 2017

The complexity of the bacterial recombination system is a barrier for the construction of bacterial mutants for the further functional investigation of specific genes. Several protocols have been developed to inactivate genes from the genus Pseudomonas. Those protocols are complicated and time-consuming and mostly do not enable easy construction of multiple knock-ins/outs. The current study describes a single and double crossover-recombination system using an optimized vector-free allele-exchange protocol for gene disruption and gene replacement in a single species of the family Pseudomonadaceae. The protocol is based on self-ligation (circularization) for the DNA cassette which has been obtained by overlapping polymerase chain reaction (Fusion-PCR), and carries an antibiotic resistance cassette flanked by homologous internal regions of the target locus. To establish the reproducibility of the approach, three different chromosomal genes (ncRNA31, rpoN, rpoS) were knocked-out from the root-associative bacterium Pseudomonas stutzeri A1501. The results showed that the P. stutzeri A1501 mutants, which are free of any plasmid backbone, could be obtained via a single or double crossover recombination. In order to optimize this protocol, three key factors that were found to have great effect on the efficiency of the homologous recombination were further investigated. Moreover, the modified protocol does not require further cloning steps, and it enables the construction of multiple gene knock-in/out mutants sequentially. This work provides a simple and rapid mutagenesis strategy for genome editing in P. stutzeri, which may also be applicable for other gram-negative bacteria.

• Reproductive and Developmental Biology
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• v.38 no.2
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• pp.71-77
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• 2014

The specific genetic modification in porcine somatic cells by gene targeting has been very difficult because of low efficiency of homologous recombination. To improve gene targeting, we designed three kinds of knock-out vectors with ${\alpha}1,3$-galactosyltransferase gene (${\alpha}1,3$-GT gene), DT-A/pGT5'/neo/pGT3', DT-A/NLS/pGT5'/neo/pGT3' and pGT5'/neo/ pGT3'/NLS. The knock-out vectors consisted of a 4.8-kb fragment as the 5' recombination arm (pGT5') and a 1.9-kb fragment as the 3' recombination arm (pGT3'). We used the neomycin resistance gene (neo) as a positive selectable marker and the diphtheria toxin A (DT-A) gene as a negative selectable marker. These vectors have a neo gene insertion in exon 9 for inactivation of ${\alpha}1,3$-GT locus. DT-A/pGT5'/neo/pGT3' vector contain only positive-negative selection marker with conventional targeting vector. DT-A/NLS/pGT5'/neo/pGT3' vector contain positive-negative selection marker and NLS sequences in upstream of 5' recombination arm which enhances nuclear transport of foreign DNA into bovine somatic cells. pGT5'/neo/pGT3'/NLS vector contain only positive selection marker and NLS sequence in downstream of 3' recombination arm, not contain negative selectable marker. For transfection, linearzed vectors were introduced into porcine ear fibroblasts by electroporation. After 48 hours, the transfected cells were selected with $300{\mu}g/ml$ G418 during 12 day. The G418-resistant colonies were picked, of which 5 colonies were positive for ${\alpha}1,3$-GT gene disruption in 3' PCR and southern blot screening. Three knock-out somatic cells were obtained from DT-A/NLS/ pGT5'/neo/pGT3' knock-out vector. Thus, these data indicate that gene targeting vector using nuclear localization signal and negative selection marker improve targeting efficiency in porcine somatic cells.

• The Transactions of The Korean Institute of Electrical Engineers
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• v.61 no.1
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• pp.85-88
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• 2012

DSCs are based on a dye-adsorbed porous $TiO_2$ layer as a photo electrode [1]. Under the illumination, dye molecules are excited and electrons are produced. The injected electrons in the conduction band of $TiO_2$ may recombine with the electrolyte. To obtain high performance DSCs, it is essential to retard the recombination. The charge recombination can be reduced by forming core-shell structure. In this work, we investigated the core-shell structure with $Al_2O_3$ and MgO coating layer on the porous $TiO_2$ layer. We confirmed the photovoltaic properties by I-V characteristics. The current and the efficiency was improved. In addition to, Through decrease in the width of EIS arc, which is the sum of the interfacial charge transfer resistances of both electrodes, we can be indicated that the block effect.

• Journal of information and communication convergence engineering
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• v.17 no.2
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• pp.161-165
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• 2019

In this study, zinc tin oxide (ZTO) thin-film transistors are researched to observe the correlation between the barrier potential and electrical properties. Although much research has been conducted on the electronic radiation from Schottky contacts in semiconductor devices, research on electronic radiation that occurs at voltages above the threshold voltage is lacking. Furthermore, the current phenomena occurring below the threshold voltage need to be studied. Bidirectional transistors exhibit current flows below the threshold voltage, and studying the characteristics of these currents can help understand the problems associated with leakage current. A factor that affects the stability of bidirectional transistors is the potential barrier to the Schottky contact. It has been confirmed that Schottky contacts increase the efficiency of the element in semiconductor devices, by cutting off the leakage current, and that the recombination at the PN junction is closely related to the Schottky contacts. The bidirectional characteristics of the transistors are controlled by the space-charge limiting currents generated by the barrier potentials of the SiOC insulated film. Space-charge limiting currents caused by the tunneling phenomenon or quantum effect are new conduction mechanisms in semiconductors, and are different from the leakage current.

• Transactions of the Korean hydrogen and new energy society
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• v.33 no.1
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• pp.113-120
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• 2022

Quantum dots (QDs) are attractive photosensitizer candidates for application not only in solar cells but also in solar hydrogen generation. For the prepartion of highly efficient QD-sensitized photoelectrodes, it is important to reduce electron recombination at the photoanode/electrolyte interface. Here, we study on the coating condition of ZnS passivation layers on the photoanodes in QD-sensitized solar cells (QDSCs). The ZnS passivation layers are coated by successive ionic layer adsorption and reaction method, and as the cation precursor, zinc acetate and zinc nitrate are empolyed. Due to the higher pH of cation precursor solution, the ZnS loading is improved when the zinc acetate is used, compared to the zinc nitrate. This improved loading of ZnS leads to the reduced electron recombination at the surface of photoanodes and the enhaced conversion efficiency of QDSCs from 6.07% to 7.45%.

• Korean Chemical Engineering Research
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• v.48 no.4
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• pp.506-510
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• 2010

Electron recombinations in electrolyte solution reduce light-to-energy conversion efficiency at the nanoporous electrode surface of dye sensitized solar cells. In this study, we improved the conversion efficiency using an energy barrier at the nanoporous electrode surface to control the recombination process. The energy barrier was formed by coating nanoporous $TiO_2$ electrode with $Nb_2O_5$ material. We investigated the influence of energy barrier on the cell efficiency depending on the coating thickness. Nanoporous $TiO_2$ electrode was coated about 5 nm thickness by 12 times coatings, and so the coating layer was grown about 0.417 nm for every time. Enhancement of conversion efficiency from 2.55% to 4.25% was achieved at 0.834 nm coating thickness, and it was believed as the optimum thickness for minimizing the electron recombination process in our experimental system.

• Journal of Information Display
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• v.3 no.2
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• pp.6-12
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• 2002

In order to achieve white emission from organic light emitting devices (OLEDs), five distinct structures were fabricated and tested. The white emission was obtained using two different color-emitting materials (yellow from rubrene-doped $Alq_3$ and blue from DPVBi) with or without a carrier-blocking layer. For enhancing the red emission, two types of devices with three-color emitting materials were fabricated. The white emission, close to the CIE coordinate of (0.3,0.3), was achieved by using two blocking layers as well that as without a blocking layer. This paper covers the subject of controlling the location of exciton recombination zone. It has been found that there is a trade-off in that the devices with three color emitting layers do not show as much luminescence efficiency compared to those with two color emitting layers, but rather, show distinct red emission in the resultant emission spectra. The highest power efficiency was measured to be 1.15lm/W at 2,000 $cd/m^2$ for a structure with two color-emitting layers.

• Journal of the Korean Institute of Electrical and Electronic Material Engineers
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• v.34 no.1
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• pp.73-77
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• 2021

Inorganic-organic hybrid perovskite solar cells have demonstrated considerable improvements, reaching 25.5% of certified power conversion efficiency in 2020 from 3.8% in 2009. In normal structured perovskite solar cells, TiO2 electron-transporting materials require heat treatment process at a high temperature over 450℃ to induce crystallinity. Inverted perovskite solar cells have also been studied to exclude the additional thermal process by using [6,6]-phenyl-C61-butyric acid methyl ester (PCBM) as a non-oxide electron-transporting layer. However, the drawback of the PCBM layer is a charge accumulation at the interface between PCBM and a metal electrode. The impact of bathocuproin (BCP) buffer layer on photovoltaic performance has been investigated herein to solve the problem of PCBM. 2-mM BCP-modified perovskite solar cells were observed to exhibit a maximum efficiency of 12.03% compared with BCP-free counterparts (5.82%) due to the suppression of the charge accumulation at the PCBM-Au interface and the resulting reduction of the charge recombination between perovskite and the PCBM layer.