• International Journal of Industrial Entomology and Biomaterials
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• v.37 no.1
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• pp.21-28
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• 2018

CRISPR/Cas9 gene editing system is an efficient method to mutation in a sequence specific manner. Here we report the direct transfection of the Cas9 nuclease and gene specific guide RNA can be used in BM-N cell line derived from Bombyx mori ovarian tissue to enfeeble function of endogenous gene in vitro. We have used gene editing system to negative regulation components of major signaling cascade, the Toll pathway, which controls B. mori resistance to microbe infections, such as fungi and gram positive bacteria. We demonstrate that the $I{\kappa}B-like$ protein Cactus may controls the activation of transcription factors such as Rel A and Rel B. The direct transfection of Cas9 nuclease and Cactus-specific guide-RNA complex may be used in BM-N cells to disrupt the function of endogenous genes in vitro. A mutation frequency of 30-40% was observed in the transfected cells, and various mutations caused the target region. Moreover, RT-PCR analysis revealed that Cactus gene was down regulated after these mutations. More importantly, mutation of BmCactus stimulated expression of lysozyme, moricin, and lebocin genes. These results suggest that the CRISPR/Cas9 systems are expected to efficiently induce site-specific mutations and it was possible to produce antimicrobial peptide through the gene editing.

• Journal of Embryo Transfer
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• v.29 no.4
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• pp.339-344
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• 2014

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein (Cas9) system can be applied to produce transgenic pigs. Therefore, we applied CRISPR/Cas9 system to generate FoxN1-targeted pig parthenogenetic embryos. Using single guided RNA targeted to pig FoxN1 genes was injected into cytoplasm of in vitro matured oocyte before electrical activation. In results, regardless of the concentrations of vector, the cleavage rate were significantly (p<0.05) decreased ($4ng/{\mu}l$, 51.24%; $8ng/{\mu}l$, 40.88%; and $16ng/{\mu}l$; 45.22%) compared to no injection group (70.44%). The blastocyst formation rates were also decreased in vector injected 3 groups ($4ng/{\mu}l$, 7.96%; $8ng/{\mu}l$, 6.4%; and $16ng/{\mu}l$; 9.04%) compared to no injection group (29.07%). In addition, the blastocyst formation rates between sham injected group (13.51%) and no injection group (29.07%) also showed significant difference (p<0.05). The mutation rates were comparable between groups ($4ng/{\mu}l$, 18.4%; $8ng/{\mu}l$, 12.5%; and $16ng/{\mu}l$; 20.0%). The sequencing analysis showed that blastocysts derived from each group were successfully mutated in FoxN1 loci regardless of the vector concentrations. However, the deletion patterns were higher than the patterns of point mutation and insertion regardless of the vector concentrations. In conclusion, we described that cytoplasmic microinjection of FoxN1-targeted CRISPR/Cas9 vector could efficiently generate transgenic pig parthenogenetic embryos in one-step.

• Development and Reproduction
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• v.23 no.3
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• pp.285-295
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• 2019

Junction-mediating and regulatory protein (JMY) is a regulator of both transcription and actin filament assembly. The actin-regulatory activity of JMY is based on a cluster of three actin-binding Wiskott-Aldrich syndrome protein homology 2 (WH2) domains that nucleate actin filaments directly and promote nucleation of the Arp2/3 complex. In addition to these activities, we examined the activity of JMY generation in early embryo of mice carrying mutations in the JMY gene by CRISPR/Cas9 mediated genome engineering. We demonstrated that JMY protein shuttled expression between the cytoplasm and the nucleus. Knockout of exon 2, CA (central domain and Arp2/3-binding acidic domain) and NLS-2 (nuclear localization signal domain) on the JMY gene by CRISPR/Cas9 system was effective and markedly impeded embryonic development. Additionally, it impaired transcription and zygotic genome activation (ZGA)-related genes. These results suggest that JMY acts as a transcription factor, which is essential for the early embryonic development in mice.

• IMMUNE NETWORK
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• v.23 no.6
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• pp.44.1-44.16
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• 2023

Mesenchymal stem cells (MSCs) are effective in treating autoimmune diseases and managing various conditions, such as engraftment of allogeneic islets. Additionally, autologous and HLA-matched allogeneic MSCs can aid in the engraftment of human allogeneic kidneys with or without low doses of tacrolimus, respectively. However, HLA alloantigens are problematic because cell therapy uses more HLA-mismatched allogeneic cells than autologous for convenience and standardization. In particular, HLA-mismatched MSCs showed increased Ag-specific T/B cells and reduced viability faster than HLA-matched MSCs. In CRISPR/Cas9-based cell therapy, Cas9 induce T cell activation in the recipient's immune system. Interestingly, despite their immunogenicity being limited to the cells with foreign Ags, the accumulation of HLA alloantigen-sensitized T/B cells may lead to allograft rejection, suggesting that alloantigens may have a greater scope of adverse effects than foreign Ags. To avoid alloantigen recognition, the β2-microglobulin knockout (B2MKO) system, eliminating class-I MHC, was able to avoid rejection by alloreactive CD8 T cells compared to controls. Moreover, universal donor cells in which both B2M and Class II MHC transactivator (CIITA) were knocked out was more effective in avoiding immune rejection than single KO. However, B2MKO and CIITA KO system remain to be controlled and validated for adverse effects such as the development of tumorigenicity due to deficient Ag recognition by CD8 T and CD4 T cells, respectively. Overall, better HLA-matching or depletion of HLA alloantigens prior to cell therapy can reduce repetitive transplantation through the long-term survival of allogeneic cell therapy, which may be especially important for patients seeking allogeneic transplantation.

• Journal of Microbiology and Biotechnology
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• v.27 no.10
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• pp.1855-1866
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• 2017

The synergistic activation mediator (SAM) system can robustly activate endogenous gene expression by a single-guide RNA. This transcriptional modulation has been shown to enhance gene promoter activity and leads to epigenetic changes. Human $interferon-{\gamma}$ is a common natural glycoprotein involved in antiviral effects and inhibition of cancer cell growth. Large quantities of high-purity $interferon-{\gamma}$ are important for medical research and clinical therapy. To investigate the possibility of employing the SAM system to enhance endogenous human $interferon-{\gamma}$ with normal function in innate immunity, we designed 10 single-guide RNAs that target 200 bp upstream of the transcription start sites of the $interferon-{\gamma}$ genome, which could significantly activate the $interferon-{\gamma}$ promoter reporter. We confirmed that the system can effectively and highly activate $interferon-{\gamma}$ expression in several humanized cell lines. Moreover, we found that the $interferon-{\gamma}$ induced by the SAM system could inhibit tumorigenesis. Taken together, our results reveal that the SAM system can modulate epigenetic traits of non-immune cells through activating $interferon-{\gamma}$ expression and triggering JAK-STAT signaling pathways. Thus, this strategy could offer a novel approach to inhibit tumorigenesis without using exogenous $interferon-{\gamma}$.

• The Korean Journal of Physiology and Pharmacology
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• v.22 no.4
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• pp.457-465
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• 2018

The expression of BCL-2 interacting cell death suppressor (BIS), an anti-stress or anti-apoptotic protein, has been shown to be regulated at the transcriptional level by heat shock factor 1 (HSF1) upon various stresses. Recently, HSF1 was also shown to bind to BIS, but the significance of these protein-protein interactions on HSF1 activity has not been fully defined. In the present study, we observed that complete depletion of BIS using a CRISPR/Cas9 system in A549 non-small cell lung cancer did not affect the induction of heat shock protein (HSP) 70 and HSP27 mRNAs under various stress conditions such as heat shock, proteotoxic stress, and oxidative stress. The lack of a functional association of BIS with HSF1 activity was also demonstrated by transient downregulation of BIS by siRNA in A549 and U87 glioblastoma cells. Endogenous BIS mRNA levels were significantly suppressed in BIS knockout (KO) A549 cells compared to BIS wild type (WT) A549 cells at the constitutive and inducible levels. The promoter activities of BIS and HSP70 as well as the degradation rate of BIS mRNA were not influenced by depletion of BIS. In addition, the expression levels of the mutant BIS construct, in which 14 bp were deleted as in BIS-KO A549 cells, were not different from those of the WT BIS construct, indicating that mRNA stability was not the mechanism for autoregulation of BIS. Our results suggested that BIS was not required for HSF1 activity, but was required for its own expression, which involved an HSF1-independent pathway.

• Journal of Veterinary Science
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• v.22 no.3
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• pp.36.1-36.12
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• 2021

Background: Mouse hepatitis virus (MHV) A59 is a highly infectious pathogen and starts in the respiratory tract and progresses to systemic infection in laboratory mice. The complement system is an important part of the host immune response to viral infection. It is not clear the role of the classical complement pathway in MHV infection. Objectives: The purpose of this study was to determine the importance of the classical pathway in coronavirus pathogenesis by comparing C1qa KO mice and wild-type mice. Methods: We generated a C1qa KO mouse using CRISPR/Cas9 technology and compared the susceptibility to MHV A59 infection between C1qa KO and wild-type mice. Histopathological and immunohistochemical changes, viral loads, and chemokine expressions in both mice were measured. Results: MHV A59-infected C1qa KO mice showed severe histopathological changes, such as hepatocellular necrosis and interstitial pneumonia, compared to MHV A59-infected wild-type mice. Virus copy numbers in the olfactory bulb, liver, and lungs of C1qa KO mice were significantly higher than those of wild-type mice. The increase in viral copy numbers in C1qa KO mice was consistent with the histopathologic changes in organs. These results indicate that C1qa deficiency enhances susceptibility to MHV A59 systemic infection in mice. In addition, this enhanced susceptibility effect is associated with dramatic elevations in spleen IFN-γ, MIP-1 α, and MCP-1 in C1qa KO mice. Conclusions: These data suggest that C1qa deficiency enhances susceptibility to MHV A59 systemic infection, and activation of the classical complement pathway may be important for protecting the host against MHV A59 infection.