The availability of genome sequences of numerous organisms and the revolution brought about by genome editing tools (e.g., ZFNs, TALENs, and CRISPR/Cas9 or RGENs) has provided a breakthrough in introducing targeted genetic changes both to... more
The availability of genome sequences of numerous organisms and the revolution brought about by genome editing tools (e.g., ZFNs, TALENs, and CRISPR/Cas9 or RGENs) has provided a breakthrough in introducing targeted genetic changes both to explore emergent phenotypes and to introduce new functionalities. However, the wider application of these tools in biology, agriculture, medicine, and biotechnology is limited by off-target mutation effects. In this review, we compare available methods for detecting, measuring, and analyzing off-target mutations. Furthermore, we particularly focus on CRISPR/Cas9 regarding various methods, tweaks, and software tools available to nullify off-target effects.
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Microalgae are versatile organisms capable of converting CO2, H2O, and sunlight into fuel and chemicals for domestic and industrial consumption. Thus, genetic modifications of microalgae for enhancing photosynthetic productivity, and... more
Microalgae are versatile organisms capable of converting CO2, H2O, and sunlight into fuel and chemicals for domestic and industrial consumption. Thus, genetic modifications of microalgae for enhancing photosynthetic productivity, and biomass and bio-products generation are crucial for both academic and industrial applications. However, targeted mutagenesis in microalgae with CRISPR-Cas9 is limited. Here we report, a one-step transformation of Chlamydomonas reinhardtii by the DNA-free CRISPR-Cas9 method rather than plasmids that encode Cas9 and guide RNAs. Outcome was the sequential CpFTSY and ZEP two-gene knockout and the generation of a strain constitutively producing zeaxanthin and showing improved photosynthetic productivity.
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As face recognition algorithms move from research labs to real world product, power consumption and cost become critical issues, and DSP-based implementations become more attractive. Also, “real-time” automatic personal identification... more
As face recognition algorithms move from research labs to real world product, power consumption and cost become critical issues, and DSP-based implementations become more attractive. Also, “real-time” automatic personal identification system should meet the conflicting dual requirements of accuracy and response time. In addition, it also should be user-friendly. This paper proposes a method of face recognition by the LDA
Research Interests: System Identification, Principal Component Analysis, Pattern Recognition, Face Recognition, Biometrics, and 14 moreDiscriminant Analysis, Equalization, Video Surveillance, Classification, Power Consumption, Color Image, Real Time, Histogram, Facies, Vector Space, Histogram Equalization, Internet, Facial Feature Extraction, and Response Time
Genome editing is crucial for genetic engineering of organisms for improved traits, particularly in microalgae due to the urgent necessity for the next generation biofuel production. The most advanced CRISPR/Cas9 system is simple,... more
Genome editing is crucial for genetic engineering of organisms for improved traits, particularly in microalgae due to the urgent necessity for the next generation biofuel production. The most advanced CRISPR/Cas9 system is simple, efficient and accurate in some organisms; however, it has proven extremely difficult in microalgae including the model alga Chlamydomonas. We solved this problem by delivering Cas9 ribonucleoproteins (RNPs) comprising the Cas9 protein and sgRNAs to avoid cytotoxicity and off-targeting associated with vector-driven expression of Cas9. We obtained CRISPR/Cas9-induced mutations at three loci including MAA7, CpSRP43 and ChlM, and targeted mutagenic efficiency was improved up to 100 fold compared to the first report of transgenic Cas9-induced mutagenesis. Interestingly, we found that unrelated vectors used for the selection purpose were predominantly integrated at the Cas9 cut site, indicative of NHEJ-mediated knock-in events. As expected with Cas9 RNPs, no off...
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CRISPR-Cas9 system is now widely used to edit a target genome in animals and plants. Cas9 protein derived from Streptococcus pyogenes (SpCas9) cleaves double-stranded DNA targeted by a chimeric single-guide RNA (sgRNA). For plant genome... more
CRISPR-Cas9 system is now widely used to edit a target genome in animals and plants. Cas9 protein derived from Streptococcus pyogenes (SpCas9) cleaves double-stranded DNA targeted by a chimeric single-guide RNA (sgRNA). For plant genome editing, Agrobacterium-mediated T-DNA transformation has been broadly used to express Cas9 proteins and sgRNAs under the control of CaMV 35S and U6/U3 promoter, respectively. We here developed a simple and high-throughput binary vector system to clone a 19-20 bp of sgRNA, which binds to the reverse complement of a target locus, in a large T-DNA binary vector containing an SpCas9 expressing cassette. Two-step cloning procedures - (1) annealing two target-specific oligonucleotides with overhangs specific to the AarI restriction enzyme site of the binary vector; (2) ligating the annealed oligonucleotides into the two AarI sites of the vector - facilitate the high-throughput production of the positive clones. In addition, Cas9-coding sequence and U6/U3 p...
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Network traffic such as Ethernet, Internet, World Wide Web, and TCP/UDP protocols has been extensively studied, with efforts focusing on the Poisson process and self-similarity. However, although SMTP (Simple Mail Transfer Protocol)... more
Network traffic such as Ethernet, Internet, World Wide Web, and TCP/UDP protocols has been extensively studied, with efforts focusing on the Poisson process and self-similarity. However, although SMTP (Simple Mail Transfer Protocol) occupies a significant portion of Internet traffic, it has attracted little attention. This paper shows that large-scale SMTP traffic possesses both the characteristics of the Poisson process and
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Site-directed mutagenesis of nitrate reductase genes using direct delivery of purified Cas9 protein preassembled with guide RNA produces mutations efficiently in Petunia × hybrida protoplast system. The clustered, regularly interspaced,... more
Site-directed mutagenesis of nitrate reductase genes using direct delivery of purified Cas9 protein preassembled with guide RNA produces mutations efficiently in Petunia × hybrida protoplast system. The clustered, regularly interspaced, short palindromic repeat (CRISPR)-CRISPR associated endonuclease 9 (CRISPR/Cas9) system has been recently announced as a powerful molecular breeding tool for site-directed mutagenesis in higher plants. Here, we report a site-directed mutagenesis method targeting Petunia nitrate reductase (NR) gene locus. This method could create mutations efficiently using direct delivery of purified Cas9 protein and single guide RNA (sgRNA) into protoplast cells. After transient introduction of RNA-guided endonuclease (RGEN) ribonucleoproteins (RNPs) with different sgRNAs targeting NR genes, mutagenesis at the targeted loci was detected by T7E1 assay and confirmed by targeted deep sequencing. T7E1 assay showed that RGEN RNPs induced site-specific mutations at frequencies ranging from 2.4 to 21 % at four different sites (NR1, 2, 4 and 6) in the PhNR gene locus with average mutation efficiency of 14.9 ± 2.2 %. Targeted deep DNA sequencing revealed mutation rates of 5.3-17.8 % with average mutation rate of 11.5 ± 2 % at the same NR gene target sites in DNA fragments of analyzed protoplast transfectants. Further analysis from targeted deep sequencing showed that the average ratio of deletion to insertion produced collectively by the four NR-RGEN target sites (NR1, 2, 4, and 6) was about 63:37. Our results demonstrated that direct delivery of RGEN RNPs into protoplast cells of Petunia can be exploited as an efficient tool for site-directed mutagenesis of genes or genome editing in plant systems.
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Targeted nucleases, including zinc-finger nucleases (ZFNs), transcription activator-like (TAL) effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9), have... more
Targeted nucleases, including zinc-finger nucleases (ZFNs), transcription activator-like (TAL) effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9), have provided researchers with the ability to manipulate nearly any genomic sequence in human cells and model organisms. However, realizing the full potential of these genome-modifying technologies requires their safe and efficient delivery into relevant cell types. Unlike methods that rely on expression from nucleic acids, the direct delivery of nuclease proteins to cells provides rapid action and fast turnover, leading to fewer off-target effects while maintaining high rates of targeted modification. These features make nuclease protein delivery particularly well suited for precision genome engineering. Here we describe procedures for implementing protein-based genome editing in human embryonic stem cells and primary cells. Protocols for the expression, purification and delivery of ZFN proteins, which are intrinsically cell-permeable; TALEN proteins, which can be internalized via conjugation with cell-penetrating peptide moieties; and Cas9 ribonucleoprotein, whose nucleofection into cells facilitates rapid induction of multiplexed modifications, are described, along with procedures for evaluating nuclease protein activity. Once they are constructed, nuclease proteins can be expressed and purified within 6 d, and they can be used to induce genomic modifications in human cells within 2 d.
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Editing plant genomes without introducing foreign DNA into cells may alleviate regulatory concerns related to genetically modified plants. We transfected preassembled complexes of purified Cas9 protein and guide RNA into plant protoplasts... more
Editing plant genomes without introducing foreign DNA into cells may alleviate regulatory concerns related to genetically modified plants. We transfected preassembled complexes of purified Cas9 protein and guide RNA into plant protoplasts of Arabidopsis thaliana, tobacco, lettuce and rice and achieved targeted mutagenesis in regenerated plants at frequencies of up to 46%. The targeted sites contained germline-transmissible small insertions or deletions that are indistinguishable from naturally occurring genetic variation.
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We can get the Pascal's matrix of order n by taking the first n rows of Pascal's triangle and filling in with 0's on the right. In this paper we obtain some well known combinatorial identities and a factorization of the... more
We can get the Pascal's matrix of order n by taking the first n rows of Pascal's triangle and filling in with 0's on the right. In this paper we obtain some well known combinatorial identities and a factorization of the Stirling matrix from the Pascal's matrix.
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In this paper, we propose an efficient method for improving visual quality of AR-FGS (Adaptive Reference FGS) which is adopted as a key scheme for SVC (Scalable Video Coding) or H.264 scalable extension. The standard FGS (Fine Granularity... more
In this paper, we propose an efficient method for improving visual quality of AR-FGS (Adaptive Reference FGS) which is adopted as a key scheme for SVC (Scalable Video Coding) or H.264 scalable extension. The standard FGS (Fine Granularity Scalability) adopts AR-FGS that introduces temporal prediction into FGS layer by using a high quality reference signal which is constructed by the weighted average between the base layer reconstructed imageand enhancement reference to improve the coding efficiency in the FGS layer. However, when the enhancement stream is truncated at certain bitstream position in transmission, the rest of the data of the FGS layer will not be available at the FGS decoder. Thus the most noticeable problem of using the enhancement layer in prediction is the degraded visual quality caused by drifting because of the mismatch between the reference frame used by the FGS encoder and that by the decoder. To solve this problem, we exploit the principle of cyclical block cod...
Video FRUC (Frame Rate Up Conversion) is one of the main issues that have arisen in recent years with the explosive growth of video sources and display formats in consumer electronics. Most advanced FRUC algorithms adopt an efficient... more
Video FRUC (Frame Rate Up Conversion) is one of the main issues that have arisen in recent years with the explosive growth of video sources and display formats in consumer electronics. Most advanced FRUC algorithms adopt an efficient motion interpolation technique to determine the motion vector field of interpolated frames. But, in some application areas such as post processing in receiver side, it is necessary to evaluate how well the MCI (Motion Compensated Interpolation) frame was reconstructed. In order to achieve this aim, first, this paper introduces some cost functions to estimate the reliability of a block in the MCI frame. Then, by using these functions, this paper proposes two distortion estimation models for evaluating how much noise was produced in the MCI frame. Through computer simulations, it is shown that the proposed estimation methods perform effectively in estimating the noises of the MCI frame.
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In this paper, we design and analyze performance of Exclusive-OR based FEC (Forward error correction) system to deploy SVC video transmission service over packet-loss prone IP network. In the designed system, we adopt standard compliant... more
In this paper, we design and analyze performance of Exclusive-OR based FEC (Forward error correction) system to deploy SVC video transmission service over packet-loss prone IP network. In the designed system, we adopt standard compliant Exclusive-OR based FEC scheme and apply it to be appropriate to the hierarchical layer structure of SVC video. To verify the performance of the designed Exclusive-OR based FEC system for SVC video transmission, we employ NIST-NET based transport simulator. By the SVC video transmission using the NIST-NET based simulator, we confirm the error resilient transmission performance of the designed Exclusive-OR based FEC system.
Recently, many research works are focusing on DVC (Distributed Video Coding) system for low complexity encoder. Most DVC systems need feedback channel for parity bit control to achieve the good RD performances, however, this causes the... more
Recently, many research works are focusing on DVC (Distributed Video Coding) system for low complexity encoder. Most DVC systems need feedback channel for parity bit control to achieve the good RD performances, however, this causes the system to have high decoding latency and is considered as one of the most critical problems for real implementation. In order to overcome this problem, this paper proposes an effective distributed video decoding method using adaptive LDPCA frame-based parity bit request estimation. The proposed method applies for the pixel-domain Wyner-Ziv system and exploits the statistical characteristics between adjacent LDPCA frames to estimate adaptively the parity bit request. Through computer simulations, it is shown that the proposed method achieves about 80% of latency reduction compared to the conventional no-estimation DVC system.
Recently, DVC (Distributed Video Coding) is receiving a lot of attention as one of the low complexity video encoding techniques suitable for various applications with computation-limited and/or power-limited environment, and is being... more
Recently, DVC (Distributed Video Coding) is receiving a lot of attention as one of the low complexity video encoding techniques suitable for various applications with computation-limited and/or power-limited environment, and is being actively studied for improving the coding efficiency. This paper proposes a rate-distortion based selective block encoding scheme. First, the motion information is obtained in the process of generating side information at decoder and received through the feedback channel, and then, based on this information, the proposed method performs a selective block encoding based on rate-distortion optimization. Experimental results show that the performance of the proposed scheme reaches up to 2.2 dB PSNR gain over the existing scheme. Moreover, it is shown that the complexity can be reduced by encoding parts of region considering rate-distortion cost.
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Scalable Video Coding (SVC) extension of H.264/AVC is a new video coding standard for media convergence by providing diverse videos of different spatial-temporal-quality layers with a single bitstream. Recently, real-time SVC codecs are... more
Scalable Video Coding (SVC) extension of H.264/AVC is a new video coding standard for media convergence by providing diverse videos of different spatial-temporal-quality layers with a single bitstream. Recently, real-time SVC codecs are being developed for the application areas of surveillance video and mobile video, etc. This paper presents the design and implementation of a H.264/SVC decoder based on an embedded DSP using Open SVC Decoder (OSD) which is a real-time software decoder designed for the PC environment. The implementation consists of porting C code of the OSD software from PC to DSP environment, profiling the complexity performance of OSD with further optimization, and integrating the optimized decoder into the TI Davinci EVM (Evaluation Module). 50 QCIF/CIF frames or 15 SD frames per second can be decoded with the implemented DSP-based SVC decoder.
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Recently, as one of several methods to improve the performance of DVC(Distributed Video Coding) system, many research works are focusing on the iterative refinement of side information. Most of the conventional techniques are mainly based... more
Recently, as one of several methods to improve the performance of DVC(Distributed Video Coding) system, many research works are focusing on the iterative refinement of side information. Most of the conventional techniques are mainly based on the relationship between the reconstruction level and side information, or the vector median filtering of motion vectors, but, their performance improvements are restricted. In order to overcome the performance limit of the conventional schemes, in this paper, a side information generation scheme is designed by measuring the block-cost estimation. Then, by adaptively selecting the compensation mode using the received bit-plane information, we propose a block-adaptive iterative refinement which is efficient for non-symmetric moving objects. Computer simulations show that, by using the proposed refinement method, the performance can be improved up to 0.2 dB in rate-distortion.
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In this paper, we propose an efficient layer-separable PES packetization and processing scheme for DVB-S2 satellite broadcasting service based on SVC video. Unlike the conventional single layer-based video coding such as MPEG-2, MPEG-4... more
In this paper, we propose an efficient layer-separable PES packetization and processing scheme for DVB-S2 satellite broadcasting service based on SVC video. Unlike the conventional single layer-based video coding such as MPEG-2, MPEG-4 and H.264, SVC can combine numerous number of video layers, which are aggregated to a single bitstream. Therefore, it is necessary to devise a new PES packetization scheme that can efficiently separate multiple video layers of SVC. In order to combine the layered characteristics of the SVC video and the robust channel coding capability of LDPC (Low Density Parity Check) of DVB-S2 for unequal error protection, we propose an efficient PES packetization in the transmitter side and PES packet processing scheme in the receiver side of DVB-S2. We prove the effectiveness of the proposed scheme in terms of processing speed and time delay required for processing of the separated layers of SVC video in the satellite broadcasting service.
Diacylglycerol acyltransferase-1 (DGAT1) is involved in the assembly of hepatitis C virus (HCV) by facilitating the trafficking of the HCV core protein to the lipid droplet. Here, we abrogated DGAT1 expression in Huh-7.5 cells by using... more
Diacylglycerol acyltransferase-1 (DGAT1) is involved in the assembly of hepatitis C virus (HCV) by facilitating the trafficking of the HCV core protein to the lipid droplet. Here, we abrogated DGAT1 expression in Huh-7.5 cells by using either the transcription activator-like effector nuclease (TALEN) or lentivirus vector short hairpin RNA (shRNA) and achieved complete long-term silencing of DGAT1. HCV entry was severely impaired in DGAT1-silenced Huh-7.5 cell lines, which showed markedly diminished claudin-1 (CLDN1) expression. In DGAT1-silenced cell lines, the forced expression of CLDN1 restored HCV entry, implying that the downregulation of CLDN1 is a critical factor underlying defective HCV entry. The expression of the gene coding for hepatocyte nuclear factor 4α (HNF4α) and other hepatocyte-specific genes was also reduced in DGAT1-silenced cell lines. After DGAT1 gene rescue, CLDN1 expression was preserved, and HCV entry was restored. Strikingly, after DGAT1 silencing, CLDN1 exp...
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Thousands of cis-elements in genomes are predicted to have vital functions. Although conservation, activity in surrogate assays, polymorphisms, and disease mutations provide functional clues, deletion from endogenous loci constitutes the... more
Thousands of cis-elements in genomes are predicted to have vital functions. Although conservation, activity in surrogate assays, polymorphisms, and disease mutations provide functional clues, deletion from endogenous loci constitutes the gold-standard test. A GATA-2-binding, Gata2 intronic cis-element (+9.5) required for hematopoietic stem cell genesis in mice is mutated in a human immunodeficiency syndrome. Because +9.5 is the only cis-element known to mediate stem cell genesis, we devised a strategy to identify functionally comparable enhancers ("+9.5-like") genome-wide. Gene editing revealed +9.5-like activity to mediate GATA-2 occupancy, chromatin opening, and transcriptional activation. A +9.5-like element resided in Samd14, which encodes a protein of unknown function. Samd14 increased hematopoietic progenitor levels/activity and promoted signaling by a pathway vital for hematopoietic stem/progenitor cell regulation (stem cell factor/c-Kit), and c-Kit rescued Samd14 loss-of-function phenotypes. Thus, the hematopoietic stem/progenitor cell cistrome revealed a mediator of a signaling pathway that has broad importance for stem/progenitor cell biology.
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Programmable nucleases, which include zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and RNA-guided engineered nucleases (RGENs) repurposed from the type II clustered, regularly interspaced short... more
Programmable nucleases, which include zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and RNA-guided engineered nucleases (RGENs) repurposed from the type II clustered, regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) system are now widely used for genome editing in higher eukaryotic cells and whole organisms, revolutionising almost every discipline in biological research, medicine, and biotechnology. All of these nucleases, however, induce off-target mutations at sites homologous in sequence with on-target sites, limiting their utility in many applications including gene or cell therapy. In this review, we compare methods for detecting nuclease off-target mutations. We also review methods for profiling genome-wide off-target effects and discuss how to reduce or avoid offtarget mutations.
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Direct delivery of purified Cas9 protein with guide RNA into plant cells, as opposed to plasmid-mediated delivery, displays high efficiency and reduced off-target effects. Following regeneration from edited cells, the ensuing plant is... more
Direct delivery of purified Cas9 protein with guide RNA into plant cells, as opposed to plasmid-mediated delivery, displays high efficiency and reduced off-target effects. Following regeneration from edited cells, the ensuing plant is also likely to bypass genetically modified organism (GMO) legislation as the genome editing complex is degraded in the recipient cells.
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Recently, KrF lithography has extended to 100nm technical node using various techniques and pushed ArF lithography to sub-100nm application. To enhance resolution, there are many problems to be solved, like dark erosion (dark film loss),... more
Recently, KrF lithography has extended to 100nm technical node using various techniques and pushed ArF lithography to sub-100nm application. To enhance resolution, there are many problems to be solved, like dark erosion (dark film loss), sloped profile, line edge roughness (LER), and so on. Also, thin resist film must be used to prevent pattern collapse. In general, the aspect ratio
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... FAST: A log-buffer based ftl scheme with fully associative sector translation. In The 2005 US-Korea Conference on Science, Technology, & Entrepreneurship August 2005. 11. Chanik Park , JaeyuSeo , Dongyoung Seo , Shinhan Kim ,... more
... FAST: A log-buffer based ftl scheme with fully associative sector translation. In The 2005 US-Korea Conference on Science, Technology, & Entrepreneurship August 2005. 11. Chanik Park , JaeyuSeo , Dongyoung Seo , Shinhan Kim , Bumsoo Kim, Cost-Efficient Memory ...
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ABSTRACT We have developed several COMA (Cycloolefin-maleic anhydride) type resists and demonstrated their good lithographic performances, especially in the isolated line. Our resist (DHA-H110) was newly upgraded for the manufacturing of... more
ABSTRACT We have developed several COMA (Cycloolefin-maleic anhydride) type resists and demonstrated their good lithographic performances, especially in the isolated line. Our resist (DHA-H110) was newly upgraded for the manufacturing of sub-100nm device in terms of bulk slope, LER (Line Edge Roughness), CD Linearity, and matching with substrate to prevent pattern collapse. The chemical structure of base resin was almost unchanged. The bulk slope resulted from high absorbency of the matrix resin was successfully overcome by introducing new additive, S1, which is an agent to remove not only top loss but also footing in the bottom. In real device application, DHA-H110 exhibits better adhesion and smaller LER than acrylate type resists on organic BARC. In addition, it shows superior pattern profile after etch process to acrylate type resists. In this paper, we suggest resist related issues for sub-100nm patterning and present lithographic performances of DHA-H110 in detail.