• BMB Reports
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• 제48권12호
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• pp.643-644
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• 2015

In eukaryotes, small RNAs play important roles in both gene regulation and resistance to viral infection. Argonaute proteins have been identified as a key component of the effector complexes of various RNA-silencing pathways, but the mechanistic roles of Argonaute proteins in these pathways are not clearly understood. To address this question, we performed single-molecule fluorescence experiments using an RNA-induced silencing complex (core-RISC) composed of a small RNA and human Argonaute 2. We found that target binding of core-RISC starts at the seed region of the guide RNA. After target binding, four distinct reactions followed: target cleavage, transient binding, stable binding, and Argonaute unloading. Target cleavage required extensive sequence complementarity and accelerated core-RISC dissociation for recycling. In contrast, the stable binding of core-RISC to target RNAs required seed-match only, suggesting a potential explanation for the seed-match rule of microRNA (miRNA) target selection.

• 대한의생명과학회지
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• 제25권4호
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• pp.372-380
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• 2019

Mice with specific modified genes are useful means of studying development and disease. The CRISPR/Cas9 system is a very powerful and effective tool for generating genetically modified mice in a simple and fast manner. To generate human IgG light kappa constant knock-in mice, we tested by microinjection of a mixture of Cas9 protein, single-guide RNA and target homologous recombinant donor DNA into zygotes. We found that the injection of 10 ng/μL of Cas9 protein and crRNA/tracrRNA, rather than single guide RNA, induced the production of knock-in mice more effectively. Thus, our study provides valuable information that will help to improve the production of knock-in mice and contribute the successful generation of humanized Ab-producing mice in Korea.

• Genomics & Informatics
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• 제19권4호
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• pp.47.1-47.12
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• 2021

Kaposi's sarcoma-associated herpesvirus (KSHV) is one of the few human oncogenic viruses, which causes a variety of malignancies, including Kaposi's sarcoma, multicentric Castleman disease, and primary effusion lymphoma, particularly in human immunodeficiency virus patients. The currently available treatment options cannot always prevent the invasion and dissemination of this virus. In recent times, siRNA-based therapeutics are gaining prominence over conventional medications as siRNA can be designed to target almost any gene of interest. The ORF57 is a crucial regulatory protein for lytic gene expression of KSHV. Disruption of this gene translation will inevitably inhibit the replication of the virus in the host cell. Therefore, the ORF57 of KSHV could be a potential target for designing siRNA-based therapeutics. Considering both sequence preferences and target site accessibility, several online tools (i-SCORE Designer, Sfold web server) had been utilized to predict the siRNA guide strand against the ORF57. Subsequently, off-target filtration (BLAST), conservancy test (fuzznuc), and thermodynamics analysis (RNAcofold, RNAalifold, and RNA Structure web server) were also performed to select the most suitable siRNA sequences. Finally, two siRNAs were identified that passed all of the filtration phases and fulfilled the thermodynamic criteria. We hope that the siRNAs predicted in this study would be helpful for the development of new effective therapeutics against KSHV.

• Molecules and Cells
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• 제44권12호
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• pp.911-919
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• 2021

The virus-induced genome editing (VIGE) system aims to induce targeted mutations in seeds without requiring any tissue culture. Here, we show that tobacco rattle virus (TRV) harboring guide RNA (gRNA) edits germ cells in a wild tobacco, Nicotiana attenuata, that expresses Streptococcus pyogenes Cas9 (SpCas9). We first generated N. attenuata transgenic plants expressing SpCas9 under the control of 35S promoter and infected rosette leaves with TRV carrying gRNA. Gene-edited seeds were not found in the progeny of the infected N. attenuata. Next, the N. attenuata ribosomal protein S5 A (RPS5A) promoter fused to SpCas9 was employed to induce the heritable gene editing with TRV. The RPS5A promoter-driven SpCas9 successfully produced monoallelic mutations at three target genes in N. attenuata seeds with TRV-delivered guide RNA. These monoallelic mutations were found in 2%-6% seeds among M₁ progenies. This editing method provides an alternative way to increase the heritable editing efficacy of VIGE.

• 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.

• Genomics & Informatics
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• 제5권1호
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• pp.6-9
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• 2007

An RNase P ribozyme library has been developed as a tool for functional genomics studies. Each clone of this library contains a random 18-mer and the sequence of M1 RNA, the catalytic subunit of RNase P. Repression of target gene expression is thus achieved by the complementary binding of mRNA to the random guide sequence and the successive target cleavage via M1 RNA. Cellular expression of the ribozyme expression was confirmed, and EGFP mRNA was used as a model to demonstrate that the RNase P ribozyme expression system can inhibit the target gene expression. The constructed RNase P ribozyme library has a complexity of $1.4\times10^7$. This novel library system should become a useful in functional genomics, to identify novel gene functions in mammalian cells.

• BMB Reports
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• 제50권1호
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• pp.20-24
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• 2017

Clustered regularly-interspaced short palindromic repeats (CRISPR) is a new and effective genetic editing tool. CRISPR was initially found in bacteria to protect it from virus invasions. In the first step, specific DNA strands of virus are identified by guide RNA that is composed of crRNA and tracrRNA. Then RNAse III is required for producing crRNA from pre-crRNA. In The second step, a crRNA:tracrRNA:Cas9 complex guides RNase III to cleave target DNA. After cleavage of DNA by CRISPR-Cas9, DNA can be fixed by Non-Homologous End Joining (NHEJ) and Homology Directed Repair (HDR). Whereas NHEJ is simple and random, HDR is much more complex and accurate. Gene editing by CRISPR is able to be applied to various biological field such as agriculture and treating genetic diseases in human.

• Asian Pacific Journal of Cancer Prevention
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• 제14권4호
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• pp.2265-2268
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• 2013

Several lines of evidence indicate that cancer is a multistep process. To survey the mechanisms involving gene alteration and miRNAs in adrenocortical cancer, we focused on transcriptional factors as a point of penetration to build a regulatory network. We derived three level networks: differentially expressed; related; and global. A topology network ws then set up for development of adrenocortical cancer. In this network, we found that some pathways with differentially expressed elements (genetic and miRNA) showed some self-adaption relations, such as EGFR. The differentially expressed elements partially uncovered mechanistic changes for adrenocortical cancer which should guide medical researchers to further achieve pertinent research.

• Journal of Plant Biotechnology
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• 제48권1호
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• pp.34-43
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• 2021

CRISPR/Cas9에 의한 유전자 교정 기술은 유용 형질을 갖는 작물 및 임목의 육성에 있어 널리 사용되고 있는 핵심 기술이다. 유전자 교정 임목 육성에는 아그로박테리움에 의한 형질전환 방법이 높은 효율로 시행된 연구가 많았고 따라서 형질전환에 사용된 플라스미드 서열이 식물 유전체 안에 존재한다는 문제가 남아 있었다. 본 연구에서는 CRISPR/Cas9을 사용하여 유전자 교정 임목을 육성하는 데 기존에 알려진 벡터 도입 기술이 아닌, 단일 가닥 가이드 RNA (sgRNA)와 Cas9 단백질을 혼합하여 만든 리보핵산단백질을 현사시나무 원형질체에 도입하는 방법을 기술하였다. 염 스트레스 내성 관련 인자 PagSAP1 유전자를 표적으로 하는 3종류의 sgRNA를 디자인하고, 각 sgRNA와 Cas9 단백질을 혼합하여 만든 리보핵산단백질을 원형질체에 도입하였다. 표적화 딥시퀀싱을 통해 리보핵산단백질 형성 시 sgRNA와 Cas9 단백질을 혼합하고 일정 시간 배양하여 안정화되는 시간이 필요한 것을 확인하였다. 또한 sgRNA3의 리보핵산단백질이 sgRNA1, sgRNA2의 리보핵산단백질보다 높은 교정 효율을 보이는 것을 확인하였다. 본 실험을 통해 리보핵산단백질을 이용한 유전자 교정 기술이 임목에도 적용될 수 있음이 확인되었고, 이는 외래 유전자 없이 유전자 교정 임목을 육성하는 데 활용할 수 있을 것으로 사료된다.

• 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.