• 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.

• 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.

• 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.

• 대한의생명과학회지
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• 제26권2호
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• pp.114-119
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• 2020

Of the various types of mice used for genome editing, conditional knock-out (cKO) mice serve as an important model for studying the function of genes. cKO mice can be produced using loxP knock-in (KI) mice in which loxP sequences (34 bp) are inserted on both sides of a specific region in the target gene. These mice can be used as KO mice that do not express a gene at a desired time or under a desired condition by cross-breeding with various Cre Tg mice. Genome editing has been recently made easy by the use of third-generation gene scissors, the CRISPR-Cas9 system. However, very few laboratories can produce mice for genome editing. Here we present a more efficient method for producing loxP KI mice. This method involves the use of an HDR vector as the target vector and ssODN as the donor DNA in order to induce homologous recombination for producing loxP KI mice. On injecting 20 ng/µL of ssODN, it was observed that the target exon was deleted or loxP was inserted on only one side. However, on injecting 10 ng/µL of the target HDR vector, the insertion of loxP was observed on both sides of the target region. In the first PCR, seven mice were identified to be loxP KI mice. The accuracy of their gene sequences was confirmed through Sanger sequencing. It is expected that the loxP KI mice produced in this study will serve as an important tool for identifying the function of the target gene.