• BMB Reports
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• v.49 no.4
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• pp.201-207
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• 2016

The CRISPR-Cas system has emerged as a fascinating and important genome editing tool. It is now widely used in biology, biotechnology, and biomedical research in both academic and industrial settings. To improve the specificity and efficiency of Cas nucleases and to extend the applications of these systems for other areas of research, an understanding of their precise working mechanisms is crucial. In this review, we summarize current studies on the molecular structures and dynamic functions of type I and type II Cas nucleases, with a focus on target DNA searching and cleavage processes as revealed by single-molecule observations.

• Journal of the Korean Magnetic Resonance Society
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• v.24 no.3
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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.

• Journal of the Korean Magnetic Resonance Society
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• v.22 no.3
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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.

• Molecules and Cells
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• v.38 no.6
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• pp.475-481
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• 2015

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 off-target mutations.

• Journal of Life Science
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• v.30 no.3
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• pp.313-319
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• 2020

Recently, cattle epidemic diseases are caused by a pathogen such as a virus or bacterium. Such diseases can spread through various pathways, such as feed intake, respiration, and contact between livestock. Diagnosis based on the ELISA (Enzyme-linked immunosorbent assay) and PCR (Polymerase chain reaction) methods has limitations because these traditional diagnostic methods are time consuming assays that require multiple steps and dedicated equipment. In this review, we propose the use of the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) Cas system based on DNA and RNA levels for early point-of-care diagnosis in cattle. In the CRISPR/Cas system, Cas effectors are classified into two classes and six subtypes. The Cas effectors included in class 2 are typically Cas9 in type II, Cas12 in type V (Cas12a and Cas12b) and Cas13 in type VI (Cas13a and Cas13b). The CRISPR/Cas system uses reporter molecules that are attached to the ssDNA strands. When the Cas enzyme cuts the ssDNA, these reporters either fluoresce or change color, indicating the presence of a specific disease marker. There are several steps in the development of a CRISPR/Cas system. The first is to select the Cas enzyme depending on DNA or RNA from pathogens (viruses or bacteria). Based on that, the next step is to integrate the optimal amplification, transducing method, and signal reporter. The CRISPR/Cas system is a powerful diagnostic tool using a gene-editing method, which is faster, better, and cheaper than traditional methods. This system could be used for early diagnosis of epidemic cattle diseases and help to control their spread.

• Biomolecules & Therapeutics
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• v.26 no.3
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• pp.298-305
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• 2018

Rhomboid family member 2 gene (Rhbdf2) is an inactive homologue lacking essential catalytic residues of rhomboid intramembrane serine proteases. The protein is necessary for maturation of tumor necrosis factor-alpha ($TNF-{\alpha}$) converting enzyme, which is the molecule responsible for the release of $TNF-{\alpha}$. In this study, Rhbdf2 knockout (KO) mice were produced by CRISPR/CAS9. To see the effects of the failure of $TNF-{\alpha}$ release induced by Rhbdf2 gene KO, collagen-induced arthritis (CIA), which is the representative $TNF-{\alpha}$ related disease, was induced in the Rhbdf2 mutant mouse using chicken collagen type II. The severity of the CIA was measured by traditional clinical scores and histopathological analysis of hind limb joints. A rota-rod test and grip strength test were employed to evaluate the severity of CIA based on losses of physical functions. The results indicated that Rhbdf2 mutant mice showed clear alleviation of the clinical severity of CIA as demonstrated by the significantly lower severity indexes. Moreover, a grip strength test was shown to be useful for the evaluation of physical functional losses by CIA. Overall, the results showed that the Rhbdf2 gene has a significant effect on the induction of CIA, which is related to $TNF-{\alpha}$.