RESUMEN
Embryo implantation failures are a major challenge in reproductive medicine, but the underlying mechanism remains poorly understood. Successful implantation requires dynamic remodeling of the endometrium through integrated proliferation and differentiation of endometrial cells including luminal epithelial, glandular epithelial, and stromal cells. Conversely, their disruption causes infertility. Spatiotemporal control of transcription is required for these processes; however, the underlying epigenetic regulation is largely unknown. In this study, we examined expression data from the human endometrium during implantation and discovered that expression of the histone lysine methyltransferase KMT2D was significantly suppressed in patients with recurrent implantation failure. Further study revealed that uterine deletion of Kmt2d in mice caused infertility due to implantation failure. Morphological analysis discovered a reduction in the number of uterine glands and aberrant differentiation of the luminal and glandular epithelium into stratified phenotypes in Kmt2d knockout uteri. Administration of leukemia inhibitory factor protein, which is expressed in uterine glands and is essential for implantation, did not rescue implantation failure in Kmt2d knockout mice, suggesting that infertility was not solely due to uterine gland dysfunction. RNA sequencing analysis revealed that Kmt2d knockout uteri displayed suppressed expression of genes involved in ion homeostasis, which may affect the uterine luminal morphology. Our study suggests that KMT2D plays an essential role in facilitating successful embryo implantation by regulating the coordinated differentiation of endometrial cells, providing valuable insights into unexplained implantation failures in women.
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DNA methylation, one of the most studied epigenetic modifications, regulates many biological processes. Dysregulation of DNA methylation is implicated in the etiology of several diseases, such as cancer and imprinting diseases. Accordingly, technologies designed to manipulate DNA methylation at specific loci are considered worthwhile and many epigenome editing technologies have been developed, which were based on ZF, TALE, and CRISPR-dCas9. Here, we describe a protocol for the application of a modified dCas9-SunTag system, which increased the efficiency of targeted demethylation and gene activation at specific DNA loci. The original SunTag system consists of 10 copies of the GCN4 peptide separated by 5-amino-acid linkers. To achieve more efficient recruitment of an anti-GCN4 scFv fused to the ten-eleven (TET) 1 hydroxylase, an enzyme that demethylates DNA, we changed the linker length to 22 amino acids. Moreover, we describe the co-recruitment of TET1 and VP64 for efficient gene activation. Since we showed the manipulation of DNA methylation at specific loci and gene activation, its application could lead to its future use in the clinic.
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Sistemas CRISPR-Cas , Metilación de ADN , Humanos , Edición Génica/métodos , Regulación de la Expresión Génica , Epigénesis Genética , Oxigenasas de Función Mixta/genética , Oxigenasas de Función Mixta/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , ARN Guía de Sistemas CRISPR-Cas/genéticaRESUMEN
Knockout mice are useful tools that can provide information about the normal function of genes, including their biochemical, developmental, and physiological roles. One problem associated with the generation of knockout mice is that the loss of some genes of interest produces a lethal phenotype. Therefore, the use of conditioned knockout mice, in which genes are disrupted in specific organs, is essential for the elucidation of disease pathogenesis and the verification of drug targets. In general, conditional knockout mice are produced using the Cre/loxP system; however, the production of the large numbers of Cre/flox knockout and control mice required for analysis requires substantial time and effort. Here, we describe the generation of liver-specific conditional knockout mice via the introduction of lipid nanoparticles encapsulating Cre mRNA into the liver of floxed mice. This technique does not require the production of offspring by mating floxed mice and is therefore more convenient than the conventional method. The results presented here demonstrate that the LNP-based method enables liver-specific gene knockout in a short period of time.
RESUMEN
The precise control of endometrial receptivity is crucial for successful embryo implantation, which is strictly regulated by the ovarian steroid hormones estrogen and progesterone. Despite our improved understanding of the genetic regulation of implantation downstream of the action of hormones, we do not know much about the epigenetic regulation that occurs during early pregnancy. To investigate the role of the N6-methyladenosine (m6A) RNA modification in embryo implantation, we generated mice with conditional deletion of Mettl14, a core component of the m6A writer complex, in the uterus. These mice were infertile due to implantation failure. We showed that Mettl14-deficient uteri had aberrant upregulation of estrogen receptor α (ERα) signaling and ERα phosphorylation, but progesterone receptor (PGR) signaling was largely unaffected. Additionally, Mettl14 deletion led to abnormal activation of the innate immune pathway in Mettl14-deficient uteri. This effect was accompanied by the infiltration of immune cells, such as macrophages and dendritic cells, into the basal region of the endometrial epithelium. Methylated RNA immunoprecipitation sequencing (MeRIP-seq) showed that genes involved in the innate immune response had decreased m6A peaks in Mettl14-deficient mice. These results suggest that Mettl14 plays a crucial role in successful implantation by precisely regulating both ERα signaling and innate immunity in the uterus.
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Receptor alfa de Estrógeno , Receptores de Estrógenos , Embarazo , Femenino , Ratones , Animales , Receptor alfa de Estrógeno/genética , Receptor alfa de Estrógeno/metabolismo , Receptores de Estrógenos/metabolismo , Epigénesis Genética , Implantación del Embrión/fisiología , Útero/metabolismo , Progesterona/metabolismo , ARN/metabolismoRESUMEN
Iron metabolism is closely associated with the pathogenesis of obesity. However, the mechanism of the iron-dependent regulation of adipocyte differentiation remains unclear. Here, we show that iron is essential for rewriting of epigenetic marks during adipocyte differentiation. Iron supply through lysosome-mediated ferritinophagy was found to be crucial during the early stage of adipocyte differentiation, and iron deficiency during this period suppressed subsequent terminal differentiation. This was associated with demethylation of both repressive histone marks and DNA in the genomic regions of adipocyte differentiation-associated genes, ãincluding Pparg, which encodes PPARγ, the master regulator of adipocyte differentiation. In addition, we identified several epigenetic demethylases to be responsible for iron-dependent adipocyte differentiation, with the histone demethylase jumonji domain-containing 1A and the DNA demethylase ten-eleven translocation 2 as the major enzymes. The interrelationship between repressive histone marks and DNA methylation was indicated by an integrated genome-wide association analysis, and was also supported by the findings that both histone and DNA demethylation were suppressed by either the inhibition of lysosomal ferritin flux or the knockdown of iron chaperone poly(rC)-binding protein 2. In summary, epigenetic regulations through iron-dependent control of epigenetic enzyme activities play an important role in the organized gene expression mechanisms of adipogenesis.
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Estudio de Asociación del Genoma Completo , Hierro , Hierro/metabolismo , Metilación de ADN/genética , Epigénesis Genética , Adipocitos/metabolismo , Histona Demetilasas con Dominio de Jumonji/genética , Histona Demetilasas con Dominio de Jumonji/metabolismoRESUMEN
CRISPR/Cas9 is the genome-editing technology that is most widely used around the world. Its widespread adoption is largely due to its simplicity and ease of use. Here, we introduce the construction of vectors and genome editing of the target gene in cells using the CRISPR/Cas9 system.
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Sistemas CRISPR-Cas , Edición Génica , Sistemas CRISPR-Cas/genéticaRESUMEN
Regulating gene expression is important for basic research and therapeutic applications. The epigenome is a record of genetic modifications such as DNA methylation and histone modifications, and epigenetic changes can play a key role in modifying gene expression. With the advent of genome editing technologies, it has become possible to manipulate the epigenome of specific genomic regions to control gene expression. In particular, CRISPR-Cas9 systems have been used widely for epigenome editing due to their high efficiency, versatility, specificity, and ease of use. Here, we describe a protocol for the upregulation of specific genes using the dCas9-SunTag system.
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Sistemas CRISPR-Cas , Edición Génica , Metilación de ADN , Epigénesis Genética , Edición Génica/métodos , Regulación de la Expresión Génica , Activación TranscripcionalRESUMEN
Epigenetic regulatory mechanisms play an important role in gene silencing and genome stability; therefore, epigenetic mutations cause a variety of diseases. Analysis of the epigenome by next-generation sequencers has revealed many epigenetic mutations in various diseases such as cancer, obesity, diabetes, autism, allergies, immune diseases, and imprinting diseases. Unfortunately, it has been difficult to identify the causative epigenetic mutations because there has been no method to generate animals with target-specific epigenetic mutations. However, it has become possible to generate such animals due to the recent development of epigenome editing technology. Here, we introduce the generation of epigenome-edited mice by target-specific DNA demethylation.
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Sistemas CRISPR-Cas , Edición Génica , Animales , Desmetilación del ADN , Metilación de ADN , Epigénesis Genética , Epigenoma , Edición Génica/métodos , RatonesRESUMEN
Synergistic effects among multiple gene mutations are involved in cancer development and progression. However, developing genetically modified mouse models to analyze various combinations of mutations is extremely labor-intensive and time-consuming. To address these problems, we developed a novel method for in vivo multiplexed genome editing of the murine uterus to model human endometrial carcinoma (EMC). To do this, we injected a CRISPR-Cas9 ribonucleoprotein complex into the uterine cavity of adult female mice, followed by electroporation. Evaluation of reporter mice demonstrated that genome editing occurred specifically in uterine epithelial cells, which are the origin of EMCs. Simultaneous targeting of Pten/Trp53/Lkb1, or targeting of Pten/Lkb1 along with the Ctnnb1ΔEx3 mutation, resulted in efficient generation of invasive tumors in wild-type females within 3 months. This novel method will enable rapid and easy validation of many combinations of gene mutations that lead to endometrial carcinogenesis.
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Neoplasias Endometriales , Edición Génica , Ratones , Femenino , Humanos , Animales , Edición Génica/métodos , Sistemas CRISPR-Cas , Ribonucleoproteínas/genética , Electroporación/métodos , Neoplasias Endometriales/genéticaRESUMEN
BACKGROUND: Epigenome-edited animal models enable direct demonstration of disease causing epigenetic mutations. Transgenic (TG) mice stably expressing epigenome-editing factors exhibit dramatic and stable changes in target epigenome modifications. Successful germline transmission of a transgene from founder mice to offspring will yield a sufficient number of epigenome-edited mice for phenotypic analysis; however, if the epigenetic mutation has a detrimental phenotypic effect, it can become difficult to obtain the next generation of animals. In this case, the phenotype of founder mice must be analyzed directly. Unfortunately, current TG mouse production efficiency (TG founders per pups born) is relatively low, and improvements would increase the versatility of this technology. RESULTS: In the current study, we describe an approach to generate epigenome-edited TG mice using a combination of both the dCas9-SunTag and piggyBac (PB) transposon systems. Using this system, we successfully generated mice with demethylation of the differential methylated region of the H19 gene (H19-DMR), as a model for Silver-Russell syndrome (SRS). SRS is a disorder leading to growth retardation, resulting from low insulin-like growth factor 2 (IGF2) gene expression, often caused by epimutations at the H19-IGF2 locus. Under optimized conditions, the efficiency of TG mice production using the PB system was approximately threefold higher than that using the conventional method. TG mice generated by this system showed demethylation of the targeted DNA region and associated changes in gene expression. In addition, these mice exhibited some features of SRS, including intrauterine and postnatal growth retardation, due to demethylation of H19-DMR. CONCLUSIONS: The dCas9-SunTag and PB systems serve as a simple and reliable platform for conducting direct experiments using epigenome-edited founder mice.
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Epigenoma , ARN Largo no Codificante , Ratones , Animales , Metilación de ADN , ARN Largo no Codificante/genética , Ratones Transgénicos , Epigénesis Genética , Trastornos del Crecimiento/genéticaRESUMEN
The generation of conditional knockout mice using the Cre-loxP system is advantageous for the functional analysis of genes. Flanked by two loxP sites (floxed) mice can be directly obtained from fertilized eggs by the CRISPR/Cas9 genome editing system. We previously reported that sequential knock-in (KI) of each loxP site by electroporation (EP) at the 1- and 2-cell embryonic stages increases the number of mice with floxed alleles compared with simultaneous KI. However, EP at the 2-cell stage frequently induced blastomere fusion. These fused embryos cannot develop to term because they are tetraploidized. In this study, we examined the following three conditions to inhibit blastomere fusion by EP at the 2-cell stage: (1) hypertonic treatment, (2) Calcium (Ca2+)-free treatment, and (3) actin polymerization inhibition. Hypertonic treatment of 2-cell stage embryos prevented blastomere fusion and facilitated blastocyst development; however, KI efficiency was decreased. Ca2+-free treatment and actin polymerization inhibition by cytochalasin B (CB) reduced fusion rate, and did not have negative effects on development and KI efficiency. These results suggest that Ca2+-free and CB treatment at the 2-cell stage is effective to generate floxed mice in combination with a sequential EP method.
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Blastómeros/metabolismo , Desarrollo Embrionario/efectos de los fármacos , Ingeniería Genética/métodos , Alelos , Animales , Sistemas CRISPR-Cas/genética , Calcio/metabolismo , Fusión Celular/métodos , Citocalasina B/metabolismo , Citocalasina B/farmacología , Electroporación/métodos , Embrión de Mamíferos/embriología , Femenino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Ratones Noqueados , Cigoto/efectos de los fármacos , Cigoto/metabolismoRESUMEN
BACKGROUND: Epigenetic modifications, including DNA methylation, play an important role in gene silencing and genome stability. Consequently, epigenetic dysregulation can cause several diseases, such as cancer, obesity, diabetes, autism, and imprinting disorders. RESULTS: We validate three methods for the generation of epigenome-edited mice using the dCas9-SunTag and single-chain variable fragment-TET1 catalytic domain. We generate model mice for Silver-Russell syndrome (SRS), an imprinting disorder, by target-specific DNA demethylation in the H19 differentially methylated region. Like SRS patients, these mice show H19 upregulation and Igf2 downregulation, leading to severe intrauterine and postnatal growth retardation. CONCLUSION: This is the first report of an imprinting disease model animal generated by targeted demethylation of specific loci of the epigenome in fertilized eggs. Epigenome-edited animals are also useful for exploring the causative epimutations in epigenetic diseases.
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Modelos Animales de Enfermedad , Epigénesis Genética , Epigenoma , Ratones , Síndrome de Silver-Russell/genética , Animales , Sistemas CRISPR-Cas , Metilación de ADN , Células Madre Embrionarias/metabolismo , Epigenómica/métodos , Humanos , ARN Largo no Codificante/genética , Síndrome de Silver-Russell/diagnóstico por imagen , Cigoto/metabolismoRESUMEN
Recently, we reported PPARα-dependent DNA demethylation of the Fgf21 promoter in the postnatal mouse liver, where reduced DNA methylation is associated with enhanced gene expression after PPARα activation. However, there is no direct evidence for the effect of site-specific DNA methylation on gene expression. We employed the dCas9-SunTag and single-chain variable fragment (scFv)-TET1 catalytic domain (TET1CD) system to induce targeted DNA methylation of the Fgf21 promoter both in vitro and in vivo. We succeeded in targeted DNA demethylation of the Fgf 21 promoter both in Hepa1-6 cells and PPARα-deficient mice, with increased gene expression response to PPARα synthetic ligand administration and fasting, respectively. This study provides direct evidence that the DNA methylation status of a particular gene may determine the magnitude of the gene expression response to activation cues.
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Desmetilación del ADN , Factores de Crecimiento de Fibroblastos/genética , Animales , Sistemas CRISPR-Cas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Metilación de ADN , Epigénesis Genética , Epigenoma , Factores de Crecimiento de Fibroblastos/metabolismo , Edición Génica/métodos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , PPAR alfa/genética , PPAR alfa/metabolismo , Regiones Promotoras Genéticas/genéticaRESUMEN
Overexpression of a gene of interest is a general approach used in both basic research and therapeutic applications. However, the conventional approach involving overexpression of exogenous genes has difficulty achieving complete genome coverage, and is also limited by the cloning capacity of viral vectors. Therefore, an alternative approach would be to drive the expression of an endogenous gene using an artificial transcriptional activator. Fusion proteins of dCas9 and a transcription activation domain, such as dCas9-VP64, are widely used for activation of endogenous genes. However, when using a single sgRNA, the activation range is low. Consequently, tiling of several sgRNAs is required for robust transcriptional activation. Here we describe the screening of factors that exhibit the best synergistic activation of gene expression with TET1 in the dCas9-SunTag format. All seven factors examined showed some synergy with TET1. Among them, VP64 gave the best results. Thus, simultaneous tethering of VP64 and TET1 to a target gene using an optimized dCas9-SunTag format synergistically activates gene expression using a single sgRNA.
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Sistemas CRISPR-Cas , Ingeniería Genética/métodos , Regulación hacia Arriba , Células A549 , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Proteína 9 Asociada a CRISPR/genética , Proteína 9 Asociada a CRISPR/metabolismo , Humanos , Oxigenasas de Función Mixta/genética , Oxigenasas de Función Mixta/metabolismo , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , ARN Guía de Kinetoplastida/genética , ARN Guía de Kinetoplastida/metabolismo , Anticuerpos de Cadena Única/genética , Anticuerpos de Cadena Única/metabolismoRESUMEN
Haploinsufficiency of NSD1, which dimethylates histone H3 lysine 36 (H3K36), causes Sotos syndrome (SoS), an overgrowth syndrome. DNMT3A and DNMT3B recognizes H3K36 trimethylation (H3K36me3) through PWWP domain to exert de novo DNA methyltransferase activity and establish imprinted differentially methylated regions (DMRs). Since decrease of H3K36me3 and genome-wide DNA hypomethylation in SoS were observed, hypomethylation of imprinted DMRs in SoS was suggested. We explored DNA methylation status of 28 imprinted DMRs in 31 SoS patients with NSD1 defect and found that hypomethylation of IGF2-DMR0 and IG-DMR in a substantial proportion of SoS patients. Luciferase assay revealed that IGF2-DMR0 enhanced transcription from the IGF2 P0 promoter but not the P3 and P4 promoters. Chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) revealed active enhancer histone modifications at IGF2-DMR0, with high enrichment of H3K4me1 and H3 lysine 27 acetylation (H3K27ac). CRISPR-Cas9 epigenome editing revealed that specifically induced hypomethylation at IGF2-DMR0 increased transcription from the P0 promoter but not the P3 and P4 promoters. NSD1 knockdown suggested that NSD1 targeted IGF2-DMR0; however, IGF2-DMR0 DNA methylation and IGF2 expression were unaltered. This study could elucidate the function of IGF2-DMR0 as a DNA methylation dependent, P0 promoter-specific enhancer. NSD1 may play a role in the establishment or maintenance of IGF2-DMR0 methylation during the postimplantation period.
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Metilación de ADN , N-Metiltransferasa de Histona-Lisina/genética , Factor II del Crecimiento Similar a la Insulina/genética , Síndrome de Sotos/genética , Sistemas CRISPR-Cas , Niño , Preescolar , Elementos de Facilitación Genéticos , Epigenoma , Femenino , Eliminación de Gen , Impresión Genómica , Células HEK293 , Histonas/química , Humanos , Lactante , Recién Nacido , Lisina/química , Masculino , Fenotipo , Mutación Puntual , Regiones Promotoras GenéticasRESUMEN
To combat organ shortage in transplantation medicine, a novel strategy has been proposed to generate human organs from exogenous pluripotent stem cells utilizing the developmental mechanisms of pig embryos/foetuses. Genetically modified pigs missing specific organs are key elements in this strategy. In this study, we demonstrate the feasibility of using a genome-editing approach to generate anephrogenic foetuses in a genetically engineered pig model. SALL1 knockout (KO) was successfully induced by injecting genome-editing molecules into the cytoplasm of pig zygotes, which generated the anephrogenic phenotype. Extinguished SALL1 expression and marked dysgenesis of nephron structures were observed in the rudimentary kidney tissue of SALL1-KO foetuses. Biallelic KO mutations of the target gene induced nephrogenic defects; however, biallelic mutations involving small in-frame deletions did not induce the anephrogenic phenotype. Through production of F1 progeny from mutant founder pigs, we identified mutations that could reliably induce the anephrogenic phenotype and hence established a line of fertile SALL1-mutant pigs. Our study lays important technical groundwork for the realization of human kidney regeneration through the use of an empty developmental niche in pig foetuses.
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Animales Modificados Genéticamente , Edición Génica/métodos , Nefronas/crecimiento & desarrollo , Ingeniería de Tejidos/métodos , Factores de Transcripción/genética , Aloinjertos/provisión & distribución , Animales , Sistemas CRISPR-Cas/genética , Estudios de Factibilidad , Femenino , Desarrollo Fetal/genética , Regulación del Desarrollo de la Expresión Génica , Técnicas de Inactivación de Genes , Humanos , Trasplante de Riñón , Masculino , Mutación , Células Madre Pluripotentes/fisiología , Regeneración/fisiología , Sus scrofa , Nucleasas de los Efectores Tipo Activadores de la Transcripción/genética , Cigoto/crecimiento & desarrolloRESUMEN
DNA methylation, one of the most studied epigenetic modifications, regulates many biological processes. Dysregulation of DNA methylation is implicated in the etiology of several diseases, such as cancer and imprinting diseases. Accordingly, technologies designed to manipulate DNA methylation at specific loci are very important, and many epigenome editing technologies have been developed, based on zinc finger proteins, TALEs, and CRISPR/dCas9 targeting. We describe a protocol to induce and assess DNA demethylation on a target gene. It is based on a modification of the dCas9-SunTag system for efficient, targeted demethylation at specific DNA loci. The original SunTag system consists of ten copies of the GCN4 peptide separated by 5-amino-acid linkers. To achieve efficient recruitment of an anti-GCN4 scFv fused to the ten-eleven (TET) 1 hydroxylase, an enzyme that demethylates DNA, we changed the linker length to 22 amino acids.
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Metilación de ADN , Edición Génica/métodos , Sistemas CRISPR-Cas , Dominio Catalítico , Desmetilación del ADN , Epigénesis Genética , Humanos , Oxigenasas de Función Mixta/genética , Reacción en Cadena de la Polimerasa/métodos , ARN Guía de Kinetoplastida/genética , Dedos de ZincRESUMEN
Autism spectrum disorder (ASD) is a neurodevelopmental disorder with core symptoms that include poor social communication, restricted interests, and repetitive behaviors. Several ASD mouse models exhibit impaired social interaction, anxiety-like behavior, and elevated perseveration. Large-scale whole exome sequencing studies identified many genes putatively associated with ASD. Like chromodomain helicase DNA binding protein 8 (CHD8), the most frequently mutated gene in individuals with ASD, the candidate gene AT-rich interaction domain 1B (ARID1B) encodes a chromatin remodeling factor. Arid1b heterozygous knockout (hKO) mice exhibited ASD-like traits related to social behavior, anxiety, and perseveration, in addition to associated features reported in some cases of ASD, such as reduced weight, impaired motor coordination, and hydrocephalus. Hydrocephalus was present in 5 of 91 hKO mice, while it was not observed in wild-type littermates (0 of 188). Genome-wide gene expression patterns in Arid1b hKO mice were similar to those in ASD patients and Chd8-haploinsufficient mice, an ASD model, and to developmental changes in gene expression in fast-spiking cells in the mouse brain. Our results suggest that Arid1b haploinsufficiency causes ASD-like phenotypes in mice.
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Trastorno del Espectro Autista/genética , Proteínas de Unión al ADN/genética , Haploinsuficiencia/genética , Factores de Transcripción/genética , Animales , Trastorno del Espectro Autista/fisiopatología , Conducta Animal , Ensamble y Desensamble de Cromatina/genética , Modelos Animales de Enfermedad , Regulación de la Expresión Génica/genética , Humanos , Hidrocefalia/genética , Hidrocefalia/fisiopatología , Ratones , Ratones NoqueadosRESUMEN
Conditional knockout using Cre/lox is essential for functional analysis of genes. CRISPR/Cas in combination with two sets of guide RNAs and a single-stranded oligonucleotide enables simultaneous insertion of two lox sequences. However, this method induces double-strand breaks at two sites on the same chromosome, which causes an undesirable chromosomal deletion and reduces the flanked lox (flox) rate. To solve this problem, we investigated a method that sequentially introduces each lox sequence at the 1-cell and 2-cell embryonic stages, respectively. The sequential method was applied to both microinjection and electroporation systems. Sequential electroporation improved the flox efficiency compared with ordinary simultaneous microinjection, leading to a high yield of offspring with floxed alleles. Finally, we directly produced Cre/lox mice containing both the Cre transgene and floxed allele via sequential electroporation using Cre zygotes, which accelerated the generation of conditional knockout mice compared with the ordinary method.
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Técnicas de Inactivación de Genes/métodos , Animales , Electroporación , Ratones Noqueados , Ratones Transgénicos , MicroinyeccionesRESUMEN
Despite the importance of DNA methylation in health and disease, technologies to readily manipulate methylation of specific sequences for functional analysis and therapeutic purposes are lacking. Here we adapt the previously described dCas9-SunTag for efficient, targeted demethylation of specific DNA loci. The original SunTag consists of ten copies of the GCN4 peptide separated by 5-amino-acid linkers. To achieve efficient recruitment of an anti-GCN4 scFv fused to the ten-eleven (TET) 1 hydroxylase, which induces demethylation, we changed the linker length to 22 amino acids. The system attains demethylation efficiencies >50% in seven out of nine loci tested. Four of these seven loci showed demethylation of >90%. We demonstrate targeted demethylation of CpGs in regulatory regions and demethylation-dependent 1.7- to 50-fold upregulation of associated genes both in cell culture (embryonic stem cells, cancer cell lines, primary neural precursor cells) and in vivo in mouse fetuses.