• Molecules and Cells
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• v.39 no.10
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• pp.750-755
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• 2016

Although innate color preference of motile organisms may provide clues to behavioral biases, it has remained a longstanding question. In this study, we investigated innate color preference of zebrafish larvae. A cross maze with different color sleeves around each arm was used for the color preference test (R; red, G; green, B; blue, Y; yellow). The findings showed that 5 dpf zebrafish larvae preferred blue over other colors (B > R > G > Y). To study innate color recognition further, tyrosinase mutants were generated using CRISPR/Cas9 system. As a model for oculocutaneous albinism (OCA) and color vision impairment, tyrosinase mutants demonstrated diminished color sensation, indicated mainly by hypopigmentation of the retinal pigment epithelium (RPE). Due to its relative simplicity and ease, color preference screening using zebrafish larvae is suitable for high-throughput screening applications. This system may potentially be applied to the analysis of drug effects on larval behavior or the detection of sensory deficits in neurological disorder models, such as autism-related disorders, using mutant larvae generated by the CRISPR/Cas9 technique.

• Journal of Genetic Medicine
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• v.20 no.2
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• pp.39-45
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• 2023

As advanced sequencing technologies continue to uncover an increasing number of variants in genes associated with human genetic diseases, there is a growing demand for systematic approaches to assess the impact of these variants on human development, health, and disease. While in silico analyses have provided valuable insights, it is essential to complement these findings with model organism studies to determine the functional consequences of genetic variants in vivo. Drosophila melanogaster is an excellent genetic model for such functional studies due to its efficient genetic technologies, high gene conservation with humans, accessibility to mutant fly resources, short life cycles, and cost-effectiveness. The traditional GAL4-UAS system, allowing precise control of gene expression through binary regulation, is frequently employed to assess the effects of monoallelic variants. Recombinase medicated cassette exchange or CRISPR-Cas9-mediated GAL4 insertion within coding introns or substitution of gene body with Kozak-Gal4 result in the loss-of-function of the target gene. This GAL4 insertion strategy also enables the expression of reference complementary DNA (cDNA) or cDNA carrying genetic variants under the control of endogenous regulatory cis elements. Furthermore, the CRISPR-Cas9-directed tissue-specific knockout and cDNA rescue system provides the flexibility to investigate candidate variants in a tissue-specific and/or developmental-timing dependent manner. In this review, we will delve into the diverse genetic techniques available in Drosophila and their applications in diagnosing and studying numerous undiagnosed diseases over the past decade.

• 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 Industrial Technology
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• v.42 no.1
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• pp.29-36
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• 2022

The CRISPR system has revolutionized gene editing field. Cas9-mediated gene editing such as Indel induction or HDR enable targeted gene disruption or precise correction of mutation. Moreover, CRISPR-based new editing tools have been developed such as base editors. In this review, we focus on gene editing in human pluripotent stem cells, which is principal technique for gene correction therapy and disease modeling. Pluripotent stem cell-specific drug YM155 enabled selection of target gene-edited pluripotent stem cells. Also, we discussed base editing for treatment of congenital retina disease. Adenine base editor delivery as RNP form provide an approach for genetic disease treatment with safe and precise in vivo gene correction.

• 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.

• Molecules and Cells
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• v.40 no.11
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• pp.823-827
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• 2017

Genome editing using programmable nucleases such as CRISPR/Cas9 or Cpf1 has emerged as powerful tools for gene knock-out or knock-in in various organisms. While most genetic diseases are caused by point mutations, these genome-editing approaches are inefficient in inducing single-nucleotide substitutions. Recently, Cas9-linked cytidine deaminases, named base editors (BEs), have been shown to convert cytidine to uridine efficiently, leading to targeted single-base pair substitutions in human cells and organisms. Here, we first report on the generation of Xenopus laevis mutants with targeted single-base pair substitutions using this RNA-guided programmable deaminase. Injection of base editor 3 (BE3) ribonucleoprotein targeting the tyrosinase (tyr) gene in early embryos can induce site-specific base conversions with the rates of up to 20.5%, resulting in oculocutaneous albinism phenotypes without off-target mutations. We further test this base-editing system by targeting the tp53 gene with the result that the expected single-base pair substitutions are observed at the target site. Collectively, these data establish that the programmable deaminases are efficient tools for creating targeted point mutations for human disease modeling in Xenopus.

• Journal of Microbiology and Biotechnology
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• v.30 no.10
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• pp.1583-1591
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• 2020

CRISPR/Cpf1 has emerged as a new CRISPR-based genome editing tool because, in comparison with CRIPSR/Cas9, it has a different T-rich PAM sequence to expand the target DNA sequence. Single-base editing in the microbial genome can be facilitated by oligonucleotide-directed mutagenesis (ODM) followed by negative selection with the CRISPR/Cpf1 system. However, single point mutations aided by Cpf1 negative selection have been rarely reported in Corynebacterium glutamicum. This study aimed to introduce an amber stop codon in crtEb encoding lycopene hydratase, through ODM and Cpf1-mediated negative selection; deficiency of this enzyme causes pink coloration due to lycopene accumulation in C. glutamicum. Consequently, on using double-, triple-, and quadruple-base-mutagenic oligonucleotides, 91.5-95.3% pink cells were obtained among the total live C. glutamicum cells. However, among the negatively selected live cells, 0.6% pink cells were obtained using single-base-mutagenic oligonucleotides, indicating that very few single-base mutations were introduced, possibly owing to mismatch tolerance. This led to the consideration of various target-mismatched crRNAs to prevent the death of single-base-edited cells. Consequently, we obtained 99.7% pink colonies after CRISPR/Cpf1-mediated negative selection using an appropriate single-mismatched crRNA. Furthermore, Sanger sequencing revealed that single-base mutations were successfully edited in the 99.7% of pink cells, while only two of nine among 0.6% of pink cells were correctly edited. The results indicate that the target-mismatched Cpf1 negative selection can assist in efficient and accurate single-base genome editing methods in C. glutamicum.

• Laboraroty Animal Research
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• v.34 no.4
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• pp.166-175
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• 2018

Recombination activating gene-2 (RAG-2) plays a crucial role in the development of lymphocytes by mediating recombination of T cell receptors and immunoglobulins, and loss of RAG-2 causes severe combined immunodeficiency (SCID) in humans. Rag-2 knockout mice created using homologous recombination in ES cells have served as a valuable immunodeficient platform, but concerns have persisted on the specificity of Rag-2-related phenotypes in these animals due to the limitations associated with the genome engineering method used. To precisely investigate the function of Rag-2, we recently established a new Rag-2 knockout FVB mouse line ($Rag-2^{-/-}$) manifesting lymphopenia by employing a CRISPR/Cas9 system at Center for Mouse Models of Human Disease. In this study, we further characterized their phenotypes focusing on histopathological analysis of lymphoid organs. $Rag-2^{-/-}$ mice showed no abnormality in development compared to their WT littermates for 26 weeks. At necropsy, gross examination revealed significantly smaller spleens and thymuses in $Rag-2^{-/-}$ mice, while histopathological investigation revealed hypoplastic white pulps with intact red pulps in the spleen, severe atrophy of the thymic cortex and disappearance of follicles in lymph nodes. However, no perceivable change was observed in the bone marrow. Moreover, our analyses showed a specific reduction of lymphocytes with a complete loss of mature T cells and B cells in the lymphoid organs, while natural killer cells and splenic megakaryocytes were increased in $Rag-2^{-/-}$ mice. These findings indicate that our $Rag-2^{-/-}$ mice show systemic lymphopenia with the relevant histopathological changes in the lymphoid organs, suggesting them as an improved Rag-2-related immunodeficient model.

• Laboraroty Animal Research
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• v.34 no.4
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• pp.302-310
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• 2018

CD47 (integrin-associated protein), a multi-spanning transmembrane protein expressed in all cells including red blood cells (RBCs) and leukocytes, interacts with signal regulatory protein ${\alpha}$ ($SIRP{\alpha}$) on macrophages and thereby inhibits phagocytosis of RBCs. Recently, we generated a novel C57BL/6J CD47 knockout ($CD47^{-/-}$ hereafter) mouse line by employing a CRISPR/Cas9 system at Center for Mouse Models of Human Disease, and here report their hematological phenotypes. On monitoring their birth and development, $CD47^{-/-}$ mice were born viable with a natural male-to-female sex ratio and normally developed from birth through puberty to adulthood without noticeable changes in growth, food/water intake compared to their age and sex-matched wild-type littermates up to 26 weeks. Hematological analysis revealed a mild but significant reduction of RBC counts and hemoglobin in 16 week-old male $CD47^{-/-}$ mice which were aggravated at the age of 26 weeks with increased reticulocyte counts and mean corpuscular volume (MCV), suggesting hemolytic anemia. Interestingly, anemia in female $CD47^{-/-}$ mice became evident at 26 weeks, but splenomegaly was identified in both genders of $CD47^{-/-}$ mice from the age of 16 weeks, consistent with development of hemolytic anemia. Additionally, helper and cytotoxic T cell populations were considerably reduced in the spleen, but not in thymus, of $CD47^{-/-}$ mice, suggesting a crucial role of CD47 in proliferation of T cells. Collectively, these findings indicate that our $CD47^{-/-}$ mice have progressive hemolytic anemia and splenic depletion of mature T cell populations and therefore may be useful as an in vivo model to study the function of CD47.

• BMB Reports
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• v.51 no.7
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• pp.350-355
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• 2018

HnRNPK is a multifunctional protein that participates in chromatin remodeling, transcription, RNA splicing, mRNA stability and translation. Here, we uncovered the function of hnRNPK in regulating the proliferation and differentiation of myoblasts. hnRNPK was mutated in the C2C12 myoblast cell line using the CRISPR/Cas9 system. A decreased proliferation rate was observed in hnRNPK-mutated cells, suggesting an impaired proliferation phenotype. Furthermore, increased G2/M phase, decreased S phase and increased sub-G1 phase cells were detected in the hnRNPK-mutated cell lines. The expression analysis of key cell cycle regulators indicated mRNA of Cyclin A2 was significantly increased in the mutant myoblasts compared to the control cells, while Cyclin B1, Cdc25b and Cdc25c were decreased sharply. In addition to the myoblast proliferation defect, the mutant cells exhibited defect in myotube formation. The myotube formation marker, myosin heavy chain (MHC), was decreased sharply in hnRNPK-mutated cells compared to control myoblasts during differentiation. The deficiency in hnRNPK also resulted in the repression of Myog expression, a key myogenic regulator during differentiation. Together, our data demonstrate that hnRNPK is required for myoblast proliferation and differentiation and may be an essential regulator of myoblast function.