• Journal of Microbiology and Biotechnology
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• 제33권9호
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• pp.1228-1237
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• 2023

The CRISPR-Cas system has emerged as the most efficient genome editing technique for a wide range of cells. Delivery of the Cas9-sgRNA ribonucleoprotein complex (Cas9 RNP) has gained popularity. The objective of this study was to develop a quantitative polymerase chain reaction (qPCR)-based assay to quantify the double-strand break reaction mediated by Cas9 RNP. To accomplish this, the dextransucrase gene (dsr) from Leuconostoc citreum was selected as the target DNA. The Cas9 protein was produced using recombinant Escherichia coli BL21, and two sgRNAs were synthesized through in vitro transcription to facilitate binding with the dsr gene. Under optimized in vitro conditions, the 2.6 kb dsr DNA was specifically cleaved into 1.1 and 1.5 kb fragments by both Cas9-sgRNA365 and Cas9-sgRNA433. By monitoring changes in dsr concentration using qPCR, the endonuclease activities of the two Cas9 RNPs were measured, and their efficiencies were compared. Specifically, the specific activities of dsr365RNP and dsr433RNP were 28.74 and 34.48 (unit/㎍ RNP), respectively. The versatility of this method was also verified using different target genes, uracil phosphoribosyl transferase (upp) gene, of Bifidobacterium bifidum and specific sgRNAs. The assay method was also utilized to determine the impact of high electrical field on Cas9 RNP activity during an efficient electroporation process. Overall, the results demonstrated that the qPCR-based method is an effective tool for measuring the endonuclease activity of Cas9 RNP.

• BMB Reports
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• 제56권2호
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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.

• 한국자기공명학회논문지
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• 제24권3호
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• pp.70-76
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• 2020

The CRISPR-Cas system provides an adaptive immunity for bacteria and archaea against invading phages or foreign plasmids. In the type II CRISPR-Cas system, a single effector protein Cas9 and a guide RNA form an RNA-guided endonuclease complex that can degrade DNA targets of foreign origin. To avoid the Cas9-mediated destruction, phages evolved anti-CRISPR (Acr) proteins that neutralize the host bacterial immunity by inactivating the CRISPR-Cas system. Here we report the backbone ¹H, ¹⁵N, and ¹³C resonance assignments of AcrIIA5 that inhibits the endonuclease activity of type II-A Streptococcus thermophilus Cas9 and also Streptococcus pyogenesis Cas9 using triple resonance nuclear magnetic resonance spectroscopy. The backbone chemical shifts of AcrIIA5 predict a disordered region at the N-terminus, followed by an αββββαβββ fold.

• 한국자기공명학회논문지
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• 제22권3호
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• pp.71-75
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• 2018

The CRISPR-Cas system is the adaptive immune system in bacteria and archaea against invading phages or foreign plasmids. In the type II CRISPR-Cas system, an endonuclease Cas9 cleaves DNA targets of phages as directed by guide RNA comprising crRNA and tracrRNA. To avoid targeting and destruction by Cas9, phages employ anti-CRISPR (Acr) proteins that act against host bacterial immunity by inactivating the CRISPR-Cas system. Here we report the backbone $^1H$, $^{15}N$, and $^{13}C$ resonance assignments of AcrIIA4 that inhibits endonuclease activity of type II-A Listeria monocytogenes Cas9 and also Streptococcus pyogenesis Cas9 using triple resonance nuclear magnetic resonance spectroscopy. The secondary structures of AcrIIA4 predicted by the backbone chemical shifts show an ${\alpha}{\beta}{\beta}{\beta}{\alpha}{\alpha}$ fold, which is used to determine the solution structure.

• 한국포장학회지
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• 제29권1호
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• pp.43-49
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• 2023

본 연구에서는 CasNa/CL의 물리적 특성을 개선하기 위하여 BN을 충진제로 활용하여 CasNa/CL/BN코팅제 및 코팅지들을 제조하였다. BN의 함량변화에 따라 제조한 CasNa/CL코팅지와 CasNa/CL/BN코팅지들의 화학적 및 형태학적 특성을 분석하였고, 기계적강도, 수증기차단특성, 표면특성, 항산화특성에 대한 분석을 통해 포장소재로써 적용 가능성을 확인하였다. SEM분석결과, CasNa/CL코팅지 표면에서 핀홀 현상이 발생하는 것을 확인하였다. 하지만, BN함량이 증가함에 따라 핀홀 현상은 감소되는 경향을 보였고 표면거칠기는 증가되는 경향을 확인할 수 있었다. BN 함량이 증가함에 따라 CasNa/CL/BN코팅지들의 연신률 및 수증기차단성이 개선되는 것을 확인할 수 있었다. BN함량 증가에도 불구하고 CasNa/CL/BN코팅지들의 항산화특성은 CasNa/CL코팅지와 유사한 경향을 보임을 확인하였다. 자연유래소재인 CasNa, CL 및 BN을 활용한 코팅지의 경우 친환경 포장소재로써 활용이 가능할 것으로 판단되지만, CL 및 BN과 CasNa와의 혼화성 및 분산성 개선 방안에 대한 추가적인 연구가 필요함을 확인하였다.

• Journal of Ginseng Research
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• 제46권4호
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• pp.505-514
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• 2022

Background: The roots of Panax ginseng contain two types of tetracyclic triterpenoid saponins, namely, protopanaxadiol (PPD)-type saponins and protopanaxatiol (PPT)-type saponins. In P. ginseng, the protopanaxadiol 6-hydroxylase (PPT synthase) enzyme catalyses protopanaxatriol (PPT) production from protopanaxadiol (PPD). In this study, we constructed homozygous mutant lines of ginseng by CRISPR/Cas9-mediated mutagenesis of the PPT synthase gene and obtained the mutant ginseng root lines having complete depletion of the PPT-type ginsenosides. Methods: Two sgRNAs (single guide RNAs) were designed for target mutations in the exon sequences of the two PPT synthase genes (both PPTa and PPTg sequences) with the CRISPR/Cas9 system. Transgenic ginseng roots were generated through Agrobacterium-mediated transformation. The mutant lines were screened by ginsenoside analysis and DNA sequencing. Result: Ginsenoside analysis revealed the complete depletion of PPT-type ginsenosides in three putative mutant lines (Cr4, Cr7, and Cr14). The reduction of PPT-type ginsenosides in mutant lines led to increased accumulation of PPD-type ginsenosides. The gene editing in the selected mutant lines was confirmed by targeted deep sequencing. Conclusion: We have established the genome editing protocol by CRISPR/Cas9 system in P. ginseng and demonstrated the mutated roots producing only PPD-type ginsenosides by depleting PPT-type ginsenosides. Because the pharmacological activity of PPD-group ginsenosides is significantly different from that of PPT-group ginsenosides, the new type of ginseng mutant producing only PPD-group ginsenosides may have new pharmacological characteristics compared to wild-type ginseng. This is the first report to generate target-induced mutations for the modification of saponin biosynthesis in Panax species using CRISPR-Cas9 system.

• International Journal of Stem Cells
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• 제16권2호
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• pp.234-243
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• 2023

The recent advances in human pluripotent stem cells (hPSCs) enable to precisely edit the desired bases in hPSCs to be used for the establishment of isogenic disease models and autologous ex vivo cell therapy. The knock-in approach based on the homologous directed repair with Cas9 endonuclease, causing DNA double-strand breaks (DSBs), produces not only insertion and deletion (indel) mutations but also deleterious large deletions. On the contrary, due to the lack of Cas9 endonuclease activity, base editors (BEs) such as adenine base editor (ABE) and cytosine base editor (CBE) allow precise base substitution by conjugated deaminase activity, free from DSB formation. Despite the limitation of BEs in transition substitution, precise base editing by BEs with no massive off-targets is suggested to be a prospective alternative in hPSCs for clinical applications. Considering the unique cellular characteristics of hPSCs, a few points should be considered. Herein, we describe an updated and optimized protocol for base editing in hPSCs. We also describe an improved methodology for CBE-based C to T substitutions, which are generally lower than A to G substitutions in hPSCs.

• 한국발생생물학회지:발생과생식
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• 제27권4호
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• pp.205-211
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• 2023

INTS14/VWA9, a component of the integrator complex subunits, plays a pivotal role in regulating the fate of numerous nascent RNAs transcribed by RNA polymerase II, particularly in the biogenesis of small nuclear RNAs and enhancer RNAs. Despite its significance, a comprehensive mutation model for developmental research has been lacking. To address this gap, we aimed to investigate the expression patterns of INTS14 during zebrafish embryonic development. We generated ints14 mutant strains using the CRISPR/Cas9 system. We validated the gRNA activity by co-injecting Cas9 protein and a single guide RNA into fertilized zebrafish eggs, subsequently confirming the presence of a 6- or 9-bp deletion in the ints14 gene. In addition, we examined the two mutant alleles through PCR analysis, T7E1 assay, TA-cloning, and sequencing. For the first time, we used the CRISPR/Cas9 system to create a model in which some sequences of the ints14 gene were removed. This breakthrough opens new avenues for in-depth exploration of the role of ints14 in animal diseases. The mutant strains generated in this study can provide a valuable resource for further investigations into the specific consequences of ints14 gene deletion during zebrafish development. This research establishes a foundation for future studies exploring the molecular mechanisms underlying the functions of ints14, its interactions with other genes or proteins, and its broader implications for biological processes.

• 대한수학교육학회지:수학교육학연구
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• 제20권2호
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• pp.185-202
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• 2010

본 연구는 중등수학교육에서 도구화된 CAS에 기반한 최적화 문제해결 활동을 통해 CAS가 교육과정 내용제시 순서에 영향을 줄 수 있는지를 분석하기 위해 설계되었다. 이를 위하여 본 연구자는 CAS를 활용한 최적화 문제해결 활동을 구안하였으며 3개월간의 CAS 활용 수업 경험이 있으나 아직 미적분학을 접해 본 적이 없는 고등학교 2학년 7명을 선정하여 총 9차시의 수업을 실시하고, 수업녹화자료와 면담을 통해 학생들의 활동을 분석하였다. 분석 결과, 학생들은 CAS를 이용하여 미 학습된 교육내용인 미분과 삼차방정식, 무리방정식의 해구하기와 그래프 분석이 포함된 최적화 문제해결활동을 수준 높게 다룰 수 있는 것으로 나타나 CAS가 교육과정 내용제시 순서에 영향을 줄 수 있음을 확인할 수 있었다.

• 한국미생물·생명공학회지
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• 제49권4호
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• pp.534-542
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• 2021

CRISPR/Cas를 이용한 유전체 편집과 유전자 발현 조절을 위한 기술에서 sgRNA는 표적서열을 인식하는 역할을 한다. gal 프로모터를 표적서열로 하여 유전체 편집에 필요한 sgRNA의 표적인식서열의 길이와 유전자 발현 조절에 필요한 sgRNA의 표적인식서열의 길이를 Cas9-NG에서 체계적으로 비교하였다. 유전체 편집의 경우, sgRNA의 표적인식서열을 구성하는 20개의 뉴클레오티드에서 3개의 뉴클레오티드의 결손만을 허용하였다. 하지만, 유전자 발현 조절에는 표적인식서열에서 11개의 뉴클레오티드가 결손되어도 표적서열을 인식하고 결합할 수 있다는 것을 밝혔다. 따라서, sgRNA의 표적인식서열에서 4개 이상의 뉴클레오티드의 결손이 있는 경우에 sgRNA/Cas9-NG는 표적 DNA 서열에 특이적으로 결합을 하지만, 엔도뉴클레아제의 활성을 갖지 못하기 때문에 유전체 편집을 할 수 없는 것으로 판단된다. 이 결과는 인공전사인자 개발과 합성생물학 분야의 다양한 CRISPR 기술 발전에 도움을 줄 것이다.