A5026826873- CRISPR and Genetic Engineering
- Pluripotent Stem Cells Research
- Animal Genetics and Reproduction
- RNA Interference and Gene Delivery
- Virus-based gene therapy research
- Advanced biosensing and bioanalysis techniques
- Glycosylation and Glycoproteins Research
- Endoplasmic Reticulum Stress and Disease
- Viral Infectious Diseases and Gene Expression in Insects
- Marine Ecology and Invasive Species
- Genetics, Aging, and Longevity in Model Organisms
- Genetics and Neurodevelopmental Disorders
- Developmental Biology and Gene Regulation
- RNA and protein synthesis mechanisms
- Innovation and Socioeconomic Development
- Renal and related cancers
- Insect symbiosis and bacterial influences
- Galectins and Cancer Biology
- DNA Repair Mechanisms
- Chromosomal and Genetic Variations
- Neuroscience and Neuropharmacology Research
- Aquaculture disease management and microbiota
- Algal biology and biofuel production
- Alzheimer's disease research and treatments
- Epigenetics and DNA Methylation
Kyoto University
2024-2025
Hiroshima University
2015-2024
Life Science Institute
2023
Tosei General Hospital
2019
Higashihiroshima Medical Center
2019
Goethe University Frankfurt
1986
Highlights•A unique k-mer database was used to identify targetable regions in human genome•A dystrophin frameshift corrected using TALENs or CRISPR-sgRNAs iPSCs•Genomic integrity tests identified minimum off-target mutagenesis by the nucleases•Dystrophin protein detected myogenic differentiation iPSCsSummaryDuchenne muscular dystrophy (DMD) is a severe muscle-degenerative disease caused mutation gene. Genetic correction of patient-derived induced pluripotent stem cells (iPSCs) CRISPR-Cas9...
Genome engineering using programmable nucleases enables homologous recombination (HR)-mediated gene knock-in. However, the labour used to construct targeting vectors containing homology arms and difficulties in inducing HR some cell type organisms represent technical hurdles for application of HR-mediated knock-in technology. Here, we introduce an alternative strategy transcription activator-like effector (TALENs) clustered regularly interspaced short palindromic repeats...
CRISPR/Cas9-mediated genome editing is a next-generation strategy for genetic modifications, not only single gene targeting, but also multiple targeted mutagenesis. To make the most of multiplexity CRISPR/Cas9, we established system constructing all-in-one expression vectors containing guide RNA cassettes and Cas9 nuclease/nickase cassette. We further demonstrated successful examples targeting including chromosomal deletions in human cells using CRISPR/Cas9 constructed with our novel system....
Although the CRISPR/Cas system has enabled one-step generation of knockout mice, low success rates cassette knock-in limit its application range. Here we show that cloning-free, direct nuclear delivery Cas9 protein complex with chemically synthesized dual RNAs enables highly efficient target digestion, leading to mice carrying a functional up 50% efficiency, compared just 10% by commonly used method consisting mRNA and single guide RNA. Our cloning-free facilitates rapid accelerating genomic...
Abstract Potatoes (Solanum tuberosum) contain α-solanine and α-chaconine, two well-known toxic steroidal glycoalkaloids (SGAs). Sprouts green tubers accumulate especially high levels of SGAs. Although SGAs were proposed to be biosynthesized from cholesterol, the biosynthetic pathway for plant cholesterol is poorly understood. Here, we identify sterol side chain reductase 2 (SSR2) potato as a key enzyme in biosynthesis related Using vitro activity assays, determined that SSR2 (St SSR2)...
Abstract The CRISPR/Cas9 system provides a powerful tool for genome editing in various model organisms, including zebrafish. establishment of targeted gene-disrupted zebrafish (knockouts) is readily achieved by CRISPR/Cas9-mediated modification. Recently, exogenous DNA integration into the via homology-independent repair was reported, but this contained mutations at junctions genomic and integrated DNA. Thus, precise modification loci remains to be achieved. Here, we describe efficient,...
Transcription activator-like effector (TALE) nuclease (TALEN) is a site-specific nuclease, which can be freely designed and easily constructed. Numerous methods of constructing TALENs harboring different TALE scaffolds repeat variants have recently been reported. However, the functionalities structurally not yet compared. Here, we report on functional differences among several types targeting same loci. Using HEK293T cell-based single-strand annealing Cel-I assays, found that with...
Glomerular podocytes express proteins, such as nephrin, that constitute the slit diaphragm, thereby contributing to filtration process in kidney. development has been analyzed mainly mice, whereas analysis of human kidney minimal because limited access embryonic kidneys. We previously reported induction three-dimensional primordial glomeruli from induced pluripotent stem (iPS) cells. Here, using transcription activator–like effector nuclease-mediated homologous recombination, we generated...
Engineered endonucleases, such as zinc-finger nucleases (ZFNs), transcription activator-like effector (TALENs), and the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system, provide a powerful approach for genome editing in animals. However, microinjection of endonucleases into embryos requires high skill level, is time consuming, may cause damage to embryos. Here, we demonstrate that electroporation endonuclease mRNAs intact can induce at targeted...
Transcription activator-like effector nucleases (TALENs) have recently arisen as effective tools for targeted genome engineering. Here, we report streamlined methods the construction and evaluation of TALENs based on 'Golden Gate TALEN TAL Effector Kit' (Addgene). We diminished array vector requirements increased assembly rates using six-module concatemerization. altered architecture native protein to increase nuclease activity replaced final destination with a mammalian expression/in vitro...
Glycoproteins misfolded in the endoplasmic reticulum (ER) are subjected to ER-associated glycoprotein degradation (gpERAD) which Htm1-mediated mannose trimming from oligosaccharide Man8GlcNAc2 Man7GlcNAc2 is rate-limiting step yeast. In contrast, roles of three Htm1 homologues (EDEM1/2/3) mammalian gpERAD have remained elusive, with a key controversy being whether EDEMs function as mannosidases or lectins. We therefore conducted transcription activator-like effector nuclease–mediated gene...
The primary cilium is an antenna-like, microtubule-based organelle on the surface of most vertebrate cells for receiving extracellular information. Although cilia form in quiescent phase, ciliary disassembly occurs when re-enter proliferative phase. It was shown that a mitotic kinase, Polo-like kinase 1 (PLK1), required cell-proliferation-coupled disassembly. Here, we report kinesin superfamily protein 2A (KIF2A), phosphorylated at T554 by PLK1, exhibits microtubule-depolymerizing activity...
Hemimetabolous, or incompletely metamorphosing, insects are phylogenetically relatively basal and comprise many pests. However, the absence of a sophisticated genetic model system, targeted gene-manipulation has limited research on hemimetabolous species. Here we use zinc-finger nuclease transcription activator-like effector technologies to produce knockouts in insect Gryllus bimaculatus. Following microinjection mRNAs encoding nucleases into cricket embryos, targeting transgene endogenous...
Mutations in the NPHS1 gene, which encodes NEPHRIN, cause congenital nephrotic syndrome, resulting from impaired slit diaphragm (SD) formation glomerular podocytes. However, methods for SD reconstitution have been unavailable, thereby limiting studies field. In present study, we established human induced pluripotent stem cells (iPSCs) a patient with an missense mutation, and reproduced process using iPSC-derived kidney organoids. The mutant NEPHRIN failed to become localized on cell surface...
Tenomodulin (Tnmd) is a type II transmembrane glycoprotein predominantly expressed in tendons and ligaments. We found that scleraxis (Scx), member of the Twist-family basic helix-loop-helix transcription factors, transcriptional activator Tnmd expression tenocytes. During embryonic development, Scx preceded Tnmd. was nearly absent ligaments Scx-deficient mice generated by activator-like effector nucleases-mediated gene disruption. mRNA levels were dramatically decreased during serial...
Mouse embryonic stem cells (mESCs) are self-renewing and capable of differentiating into any the three germ layers. An interesting feature mESCs is presence cell-to-cell heterogeneity in gene expression that may be responsible for cell fate decisions. Nanog, a key transcription factor pluripotency, displays heterogeneous mESCs, via mechanisms not fully understood. To understand this variability, we quantitatively analyzed Nanog found was both infrequently transcribed pulsatile stochastic...
Transcription activator-like effector nucleases (TALENs) have become powerful tools for targeted genome editing. Here we demonstrate efficient mutagenesis in medaka (Oryzias latipes), which serves as an excellent vertebrate model genetics and genomics. We designed constructed a pair of TALENs targeting the DJ-1 gene, homolog human (PARK7). These induced number insertions deletions injected embryos with extremely high efficiency. This induction mutations occurred dose-dependent manner. All...
Chromosomal aneuploidy and specific gene mutations are recognized early hallmarks of many oncogenic processes. However, the net effect these abnormalities has generally not been explored. We focused on transient myeloproliferative disorder (TMD) in Down syndrome, which is characteristically associated with somatic GATA1. To better understand functional interplay between trisomy 21 GATA1 hematopoiesis, we constructed cellular disease models using human induced pluripotent stem cells (iPSCs)...
DEP-domain containing 5 (DEPDC5), encoding a repressor of the mechanistic target rapamycin complex 1 (mTORC1) signaling pathway, has recently emerged as major gene mutated in familial focal epilepsies and cortical dysplasia. Here we established global knockout rat using TALEN technology to investigate vivo impact Depdc5-deficiency. Homozygous Depdc5(-/-) embryos died from embryonic day 14.5 due growth delay. Constitutive mTORC1 hyperactivation was evidenced brains cultured fibroblasts...
Genome editing research of human ES/iPS cells has been accelerated by clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9) and transcription activator-like effector nucleases (TALEN) technologies. However, the efficiency biallelic genetic engineering in transcriptionally inactive genes is still low, unlike that active genes. To enhance homologous recombination cells, we performed screenings accessorial compounds. We found RAD51 overexpression valproic...