RESUMEN
Wilson's disease (WD) is a copper metabolic disorder caused by a defective ATP7B function. Conventional therapies cause severe side effects and significant variation in efficacy, according to cohort studies. Thus, exploring new therapeutic approaches to prevent progression to liver failure is urgent. To study the physiology and pathology of WD, immortalized cell lines and rodent WD models have been used conventionally; however, a large gap remains among different species as well as in genetic backgrounds among individuals. We generated induced pluripotent stem cells (iPSCs) from four WD patients carrying compound heterozygous mutations in the ATP7B gene. ATP7B loss- and gain-of-functions were further manifested with ATP7B-deficient iPSCs and heterozygously corrected R778L WD patient-derived iPSCs using CRISPR-Cas9-based gene editing. Although the expression of ATP7B protein varied among WD-specific hepatocytes differentiated from these iPSCs, the expression and secretion of ceruloplasmin (Cp), a downstream copper carrier in plasma, were consistently decreased in WD patient-derived and ATP7B-deficient hepatocytes. A transcriptome analysis detected abnormalities in the retinoid signaling pathway and lipid metabolism in WD-specific hepatocytes. Drug screening using WD patient-derived hepatocytes identified retinoids as promising candidates for rescuing Cp secretion. All-trans retinoic acid also alleviates reactive oxygen species production induced by lipid accumulation in WD-specific hepatocytes treated with oleic acid. These patient-derived iPSC-based hepatic models function as effective platforms for the development of potential therapeutics for hepatic steatosis in WD and other fatty liver diseases.
Asunto(s)
Degeneración Hepatolenticular , Humanos , Degeneración Hepatolenticular/tratamiento farmacológico , Degeneración Hepatolenticular/genética , Ceruloplasmina/genética , Ceruloplasmina/metabolismo , Cobre/metabolismo , Retinoides/metabolismo , Retinoides/uso terapéutico , ATPasas Transportadoras de Cobre/genética , Hepatocitos/metabolismo , Estrés Oxidativo , MutaciónRESUMEN
Fibroblast growth factor 5 (FGF5) is an important molecule required for the transition from anagen to catagen phase of the mammalian hair cycle. We previously reported that Syrian hamsters harboring a 1-bp deletion in the Fgf5 gene exhibit excessive hair growth in males. Herein, we generated Fgf5 mutant mice using genome editing via oviductal nucleic acid delivery (GONAD)/improved GONAD (i-GONAD), an in vivo genome editing system used to target early embryos present in the oviductal lumen, to study gender differences in hair length in mutant mice. The two lines (Fgf5go-malc), one with a 2-bp deletion (c.552_553del) and the other with a 1-bp insertion (c.552_553insA) in exon 3 of Fgf5, were successfully established. Each mutation was predicted to disrupt a part of the FGF domain through frameshift mutation (p.Glu184ValfsX128 or p.Glu184ArgfsX128). Fgf5go-malc1 mice had heterogeneously distributed longer hairs than wild-type mice (C57BL/6J). Notably, this change was more evident in males than in females (p < 0.0001). Immunohistochemical analysis revealed the presence of FGF5 protein in the dermal papilla and outer root sheath of the hair follicles from C57BL/6J and Fgf5go-malc1 mice. Histological analysis revealed that the prolonged anagen phase might be the cause of accelerated hair growth in Fgf5go-malc1 mice.
Asunto(s)
Factor 5 de Crecimiento de Fibroblastos , Cabello , Caracteres Sexuales , Animales , Femenino , Factor 5 de Crecimiento de Fibroblastos/genética , Factor 5 de Crecimiento de Fibroblastos/metabolismo , Cabello/crecimiento & desarrollo , Folículo Piloso/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Mutación , Ácidos Nucleicos/metabolismo , Factores SexualesRESUMEN
Genome editing using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) predominantly induces non-homologous end joining (NHEJ), which generates random insertions or deletions, whereas homology-directed repair (HDR), which generates precise recombination products, is useful for wider applications. However, the factors that determine the ratio of HDR to NHEJ products after CRISPR/Cas9 editing remain unclear, and methods by which the proportion of HDR products can be increased have not yet been fully established. We systematically analyzed the HDR and NHEJ products after genome editing using various modified guide RNAs (gRNAs) and Cas9 variants with an enhanced conformational checkpoint to improve the fidelity at endogenous gene loci in HEK293T cells and HeLa cells. We found that these modified gRNAs and Cas9 variants were able to enhance HDR in both single-nucleotide substitutions and a multi-kb DNA fragment insertion. Our results suggest that the original CRISPR/Cas9 system from the bacterial immune system is not necessarily the best option for the induction of HDR in genome editing and indicate that the modulation of the kinetics of conformational checkpoints of Cas9 can optimize the HDR/NHEJ ratio.
Asunto(s)
Proteína 9 Asociada a CRISPR/química , Proteína 9 Asociada a CRISPR/metabolismo , Edición Génica , Reparación del ADN por Recombinación , Proteína 9 Asociada a CRISPR/genética , ADN/metabolismo , Reparación del ADN por Unión de Extremidades , Variación Genética , Guanina , Células HEK293 , Células HeLa , Humanos , Reacción en Cadena de la Polimerasa , Conformación Proteica , ARN/químicaRESUMEN
The Foxe3rct mutation, which causes early-onset cataracts, is a recessive mutation found in SJL/J mice. A previous study reported that cataract phenotypes are modified by the genetic background of mouse inbred strains and that the Pde6brd1 mutation, which induced degeneration of the photoreceptor cells, is a strong candidate genetic modifier to accelerate the severity of cataractogenesis of Foxe3rct mice. We created congenic mice by transferring a genomic region including the Foxe3rct mutation to the B6 genetic background, which does not carry the Pde6brd1 mutation. In the congenic mice, the cataract phenotypes became remarkably mild, and the development of cataracts was suppressed for a long time. Moreover, we created transgenic mice by injecting BAC clones including the wild-type Pde6b gene into the eggs of SJL-Foxe3rct mice. Although the resistant effect for cataract phenotypes in transgenic mice was less than that in congenic mice, the severity and onset time of cataract phenotypes were clearly improved and delayed, respectively, compared with the phenotypes of the original SJL-Foxe3rct mice. These results clearly show that the development of early-onset cataracts requires at least two mutant alleles of Foxe3rct and Pde6brd1, and another modifier associated with the severity of cataract phenotypes in Foxe3rct mice underlies the genetic backgrounds in mice.
Asunto(s)
Catarata/genética , Catarata/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6/genética , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6/metabolismo , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Predisposición Genética a la Enfermedad/genética , Animales , Progresión de la Enfermedad , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Relación Estructura-ActividadRESUMEN
As a versatile genome editing tool, the CRISPR-Cas9 system induces DNA double-strand breaks at targeted sites to activate mainly two DNA repair pathways: HDR which allows precise editing via recombination with a homologous template DNA, and NHEJ which connects two ends of the broken DNA, which is often accompanied by random insertions and deletions. Therefore, how to enhance HDR while suppressing NHEJ is a key to successful applications that require precise genome editing. Histones are small proteins with a lot of basic amino acids that generate electrostatic affinity to DNA. Since H2A.X is involved in DNA repair processes, we fused H2A.X to Cas9 and found that this fusion protein could improve the HDR/NHEJ ratio by suppressing NHEJ. As various post-translational modifications of H2A.X play roles in the regulation of DNA repair, we also fused H2A.X mimicry variants to replicate these post-translational modifications including phosphorylation, methylation, and acetylation. However, none of them were effective to improve the HDR/NHEJ ratio. We further fused other histone variants to Cas9 and found that H2A.1 suppressed NHEJ better than H2A.X. Thus, the fusion of histone variants to Cas9 is a promising option to enhance precise genome editing.
Asunto(s)
Proteína 9 Asociada a CRISPR , Sistemas CRISPR-Cas , Reparación del ADN por Unión de Extremidades , Edición Génica , Histonas , Histonas/metabolismo , Histonas/genética , Humanos , Proteína 9 Asociada a CRISPR/metabolismo , Proteína 9 Asociada a CRISPR/genética , Edición Génica/métodos , Procesamiento Proteico-Postraduccional , Roturas del ADN de Doble Cadena , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/genética , Células HEK293 , AcetilaciónRESUMEN
Single-cell cloning is the simplest strategy to isolate genome-edited cell clones, although its scalability has been an issue. Here, we present a protocol to establish genome-edited human cultured cell clones using the On-chip SPiS, a single-cell auto-dispensing device with image recognition technology. Human cultured cells are transfected with plasmids of the CRISPR-Cas9 components, and Cas9-expressing cells are sorted and individually plated into multi-well plates by the On-chip SPiS. For complete details on the use and execution of this protocol, please refer to Takahashi et al. (2022).1.
Asunto(s)
Línea Celular , Humanos , Movimiento Celular , Transporte de Proteínas , Clonación MolecularRESUMEN
Even when precise nucleotide manipulations are intended, the outcomes of genome editing can be diverse, often including random insertions and deletions. The combinations and frequencies of these different outcomes in single cells are critical not only in the generation of genetically modified cell lines but also in the evaluation of the clinical effects of genome editing therapies. However, current methods only analyze cell populations, not single cells. Here, we utilized the Single Particle isolation System (SPiS) for the efficient isolation of single cells to systematically analyze genome editing results in individual human cultured cells. As a result, we discovered that genome editing induction has a binary nature, that is, the target alleles of cells tend to be all edited or not edited at all. This study enhances our understanding of the induction pattern of genome editing and provides a new strategy to analyze genome editing outcomes in single cells.
RESUMEN
The Rinshoken cataract (rct) mutation, which causes congenital cataracts, is a recessive mutation found in SJL/J mice. All mutants present with opacity in the lens by 2 months of age. The rct locus was mapped to a 1.6-Mb region in Chr 4 that contains the Foxe3 gene. This gene is responsible for cataracts in humans and mice, and it plays a crucial role in the development of the lens. Furthermore, mutation of Foxe3 causes various ocular defects. We sequenced the genomic region of Foxe3, including the coding exons and UTRs; however, no mutations were discovered in these regions. Because there were no differences in Foxe3 sequences between the rct/rct and wild-type mice, we inferred that a mutation was located in the regulatory regions of the Foxe3 gene. To test this possibility, we sequenced a 5' noncoding region that is highly conserved among vertebrates and is predicted to be the major enhancer of Foxe3. This analysis revealed a deletion of 22-bp located approximately 3.2-kb upstream of the start codon of Foxe3 in rct mice. Moreover, we demonstrated by RT-PCR and in situ hybridization that the rct mutant has reduced expression of Foxe3 in the lens during development. We therefore suggest that cataracts in rct mice are caused by reduced Foxe3 expression in the lens and that this decreased expression is a result of a deletion in a cis-acting regulatory element.
Asunto(s)
Catarata/genética , Factores de Transcripción Forkhead/genética , Cristalino/patología , Microftalmía/genética , Secuencias Reguladoras de Ácidos Nucleicos , Animales , Catarata/patología , Factores de Transcripción Forkhead/biosíntesis , Ratones , Mutación , Eliminación de SecuenciaRESUMEN
The pronuclear injection (PI)-based targeted transgenesis (PITT) method allows the generation of targeted transgenic (Tg) mice wherein a single copy of a transgene is integrated into the Rosa26 locus following PI. The Rosa26 locus allows unbiased ubiquitous expression of integrated transgenes; however, it remains little known whether tissue-specific promoters retain their functional properties when placed at the Rosa26 locus. We evaluated tissue-specific activity and reproducibility of exogenous tissue-specific promoters targeted to the Rosa26 locus by generating Thy1-Dre/Dre reporter mice using PITT and assessed spatial expression patterns of the transgenes. The Thy1 promoter targeted to the Rosa26 locus appeared active in virtually all Purkinje cells in the cerebellum and hippocampus. However, mosaic expression of the transgene under the Thy1 promoter was observed in many other organs. This phenomenon was consistent in all the Tg lines generated by PITT, indicating a high degree of reproducibility for this experiment.
Asunto(s)
Expresión Génica/genética , Regiones Promotoras Genéticas/genética , ARN no Traducido/genética , Animales , Encéfalo/metabolismo , Genotipo , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , ARN no Traducido/metabolismo , Transgenes/genéticaRESUMEN
The excision of genomic sequences using paired CRISPR-Cas nucleases is a powerful tool to study gene function, create disease models and holds promise for therapeutic gene editing. However, our understanding of the factors that favor efficient excision is limited by the lack of a rapid, accurate measurement of DNA excision outcomes that is free of amplification bias. Here, we introduce ddXR (droplet digital PCR eXcision Reporter), a method that enables the accurate and sensitive detection of excisions and inversions independent of length. The method can be completed in a few hours without the need for next-generation sequencing. The ddXR method uncovered unexpectedly high rates of large (> 20 kb) excisions and inversions, while also revealing a surprisingly low dependence on linear distance, up to 170 kb. We further modified the method to measure precise repair of excision junctions and allele-specific excision, with important implications for disease modeling and therapeutic gene editing.
Asunto(s)
Sistemas CRISPR-Cas , Inversión Cromosómica , ADN/genética , ADN/metabolismo , Edición Génica , Células Madre Pluripotentes Inducidas/fisiología , Reacción en Cadena de la Polimerasa/métodos , Células Cultivadas , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismoRESUMEN
The control of delayed emesis is very important in order to continue ambulatory chemotherapy. We performed retrospective study that examined the efficacy of preventive treatment (granisetron+dexamethasone+domperidone) for delayed emesis induced by FOLFOX4 chemotherapy for advanced and recurrent colorectal cancer. The subjects were 92 patients who underwent FOLFOX4 chemotherapy at the Cancer Institute Hospital of JFCR (group with preventive treatment: 50, group without preventive treatment: 42), and the observation period was set as the 1st-9th cycle. The complete nausea inhibition rate was 50.0% in the group with and 21.5% in the group without preventive treatment, showing a significantly higher inhibition rate in the former (p=0.0047). The complete vomiting inhibition rates were 86.0% and 66.7%, respectively, again showing a significantly higher inhibition rate in the former (p=0.015). On multivariate analysis (multiple logistic analysis), the development of nausea/vomiting and preventive treatment for delayed emesis were significantly associated, showing that the treatment was an independent preventive factor. All adverse reactions induced by preventive treatment were mild, suggesting no safety-related problem. These findings suggested the usefulness of preventive treatment with granisetron, dexamethasone, and domperidone for FOLFOX4 chemotherapy-induced delayed emesis.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Dexametasona/uso terapéutico , Domperidona/uso terapéutico , Granisetrón/uso terapéutico , Vómitos/inducido químicamente , Vómitos/prevención & control , Adulto , Anciano , Neoplasias Colorrectales/tratamiento farmacológico , Quimioterapia Combinada , Femenino , Fluorouracilo/efectos adversos , Humanos , Leucovorina/efectos adversos , Masculino , Persona de Mediana Edad , Recurrencia Local de Neoplasia , Compuestos Organoplatinos/efectos adversos , Estudios RetrospectivosRESUMEN
We have been collecting data on the adverse reaction of FOLFOX 4 chemotherapy in our hospital from April 2005 to October retrospectively, by electronic clinical records. A retrospective study of 123 patients receiving FOLFOX 4 for advanced colorectal cancer was conducted. Survey results showed high incidences of hemotoxicity (52.8%), chronic sensory neuropathy (16.2%) and allergic reactions (15.4%). In the initial FOLFOX 4 therapy, appetite loss (60.1%), vomiting (19.5%) and acute sensory neuropathy (33.3%) were observed. We prepared a brochure in order to minimize inter-individual differences in pharmaceutical care and drug consultation by clinical pharmacists and to ensure the accurate understanding of patients. We feel sure that this kind of activity will help us to provide better pharmaceutical care for patients.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Servicios de Información sobre Medicamentos , Adulto , Anciano , Neoplasias Colorrectales/tratamiento farmacológico , Femenino , Fluorouracilo/efectos adversos , Humanos , Leucovorina/efectos adversos , Masculino , Persona de Mediana Edad , Compuestos Organoplatinos/efectos adversos , Estudios RetrospectivosRESUMEN
Major intrinsic protein of lens fiber (MIP) is one of the proteins essential for maintaining lens transparency while also contributing to dominant cataracts in humans. The Nodai cataract (Nat) mice harbor a spontaneous mutation in Mip and develop early-onset nuclear cataracts. The Nat mutation is a c.631G>A mutation (MipNat), resulting in a glycine-to-arginine substitution (p.Gly211Arg) in the sixth transmembrane domain. The MipNat/Nat homozygotes exhibit congenital cataracts caused by the degeneration of lens fiber cells. MIP normally localizes to the lens fiber cell membranes. However, the MipNat/Nat mice were found to lack an organelle-free zone, and the MIP was mislocalized to the nuclear membrane and perinuclear region. Furthermore, the MipNat/+ mice exhibited milder cataracts than MipNat/Nat mice due to the slight degeneration of the lens fiber cells. Although there were no differences in the localization of MIP to the membranes of lens fiber cells in MipNat/+ mice compared to that in wild-type mice, the protein levels of MIP were significantly reduced in the eyes. These findings suggest that cataractogenesis in MipNat mutants are caused by defects in MIP expression. Overall, the MipNat mice offer a novel model to better understand the phenotypes and mechanisms for the development of cataracts in patients that carry missense mutations in MIP.
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Acuaporinas/deficiencia , Acuaporinas/genética , Catarata/genética , Proteínas del Ojo/genética , Estudios de Asociación Genética , Mutación Missense/genética , Sustitución de Aminoácidos/genética , Animales , Acuaporinas/química , Acuaporinas/fisiología , Arginina , Proteínas del Ojo/química , Proteínas del Ojo/fisiología , Glicina , Humanos , Ratones Endogámicos BALB C , Ratones EndogámicosRESUMEN
Transgenic technologies used for creating a desired genomic change in animals involve three critical steps: isolation of fertilized eggs, microinjection of transgenic DNA into them and their subsequent transfer to recipient females. These ex vivo steps have been widely used for over 3 decades and they were also readily adapted for the latest genome editing technologies such as ZFNs, TALENs, and CRISPR/Cas9 systems. We recently developed a method called GONAD (Genome editing via Oviductal Nucleic Acids Delivery) that does not require all the three critical steps of transgenesis and therefore relieves the bottlenecks of widely used animal transgenic technologies. Here we provide protocols for the GONAD system.
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Sistemas CRISPR-Cas , Embrión de Mamíferos/metabolismo , Ingeniería Genética/métodos , Genoma/genética , Oviductos/metabolismo , Animales , Electroporación/métodos , Femenino , Técnicas de Transferencia de Gen , Ratones Noqueados , Ratones Transgénicos , Microinyecciones , Reproducibilidad de los Resultados , Cigoto/metabolismoRESUMEN
Microinjection is considered the gold standard technique for delivery of nucleic acids (NAs; transgenes or genome editing tools such as CRISPR/Cas9 systems) into embryos, for creating genetically modified organisms. It requires sophisticated equipment as well as well-trained and highly skilled personnel to perform the micro-injection technique. Here, we describe a novel and simple microinjection-independent technique, called Genome-editing via Oviductal Nucleic Acids Delivery (GONAD). Using GONAD, we show that NAs (e.g., eGFP mRNA or Cas9 mRNA/sgRNAs) can be effectively delivered to pre-implantation embryos within the intact mouse oviduct by a simple electroporation method, and result in the desired genetic modification in the embryos. Thus GONAD can bypass many complex steps in transgenic technology such as isolation of zygotes, microinjection of NAs into them, and their subsequent transfer to pseudo-pregnant animals. Furthermore, this method can potentially be used for genome editing in species other than mice.
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Electroporación/métodos , Marcación de Gen/métodos , Ingeniería Genética/métodos , Microinyecciones/métodos , Animales , Sistemas CRISPR-Cas/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Quinasa del Factor 2 de Elongación/genética , Embrión de Mamíferos/citología , Femenino , Proteínas Fluorescentes Verdes/genética , Hipoxantina Fosforribosiltransferasa/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Oviductos/citología , ARN Mensajero/genéticaRESUMEN
The availability of techniques to create desired genetic mutations has enabled the laboratory mouse as an extensively used model organism in biomedical research including human genetics. A new addition to this existing technical repertoire is the CRISPR/Cas system. Specifically, this system allows editing of the mouse genome much more quickly than the previously used techniques, and, more importantly, multiple mutations can be created in a single experiment. Here we provide protocols for preparation of CRISPR/Cas reagents and microinjection into one-cell mouse embryos to create knockout or knock-in mouse models.
Asunto(s)
Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Genoma , Animales , RatonesRESUMEN
Microphthalmia is a severe ocular disorder, and this condition is typically caused by mutations in transcription factors that are involved in eye development. Mice carrying mutations in these transcription factors would be useful tools for defining the mechanisms underlying developmental eye disorders. We discovered a new spontaneous recessive microphthalmos mouse mutant in the Japanese wild-derived inbred strain KOR1/Stm. The homozygous mutant mice were histologically characterized as microphthalmic by the absence of crystallin in the lens, a condition referred to as aphakia. By positional cloning, we identified the nonsense mutation c.444C>A outside the genomic region that encodes the homeodomain of the paired-like homeodomain transcription factor 3 gene (Pitx3) as the mutation responsible for the microphthalmia and aphakia. We examined Pitx3 mRNA expression of mutant mice during embryonic stages using RT-PCR and found that the expression levels are higher than in wild-type mice. Pitx3 over-expression in the lens during developmental stages was also confirmed at the protein level in the microphthalmos mutants via immunohistochemical analyses. Although lens fiber differentiation was not observed in the mutants, strong PITX3 protein signals were observed in the lens vesicles of the mutant lens. Thus, we speculated that abnormal PITX3, which lacks the C-terminus (including the OAR domain) as a result of the nonsense mutation, is expressed in mutant lenses. We showed that the expression of the downstream genes Foxe3, Prox1, and Mip was altered because of the Pitx3 mutation, with large reductions in the lens vesicles in the mutants. Similar profiles were observed by immunohistochemical analysis of these proteins. The expression profiles of crystallins were also altered in the mutants. Therefore, we speculated that the microphthalmos/aphakia in this mutant is caused by the expression of truncated PITX3, resulting in the abnormal expression of downstream targets and lens fiber proteins.