RESUMEN
Tardigrades are microscopic animals renowned for their ability to withstand extreme conditions, including high doses of ionizing radiation (IR). To better understand their radio-resistance, we first characterized induction and repair of DNA double- and single-strand breaks after exposure to IR in the model species Hypsibius exemplaris. Importantly, we found that the rate of single-strand breaks induced was roughly equivalent to that in human cells, suggesting that DNA repair plays a predominant role in tardigrades' radio-resistance. To identify novel tardigrade-specific genes involved, we next conducted a comparative transcriptomics analysis across three different species. In all three species, many DNA repair genes were among the most strongly overexpressed genes alongside a novel tardigrade-specific gene, which we named Tardigrade DNA damage Response 1 (TDR1). We found that TDR1 protein interacts with DNA and forms aggregates at high concentration suggesting it may condensate DNA and preserve chromosome organization until DNA repair is accomplished. Remarkably, when expressed in human cells, TDR1 improved resistance to Bleomycin, a radiomimetic drug. Based on these findings, we propose that TDR1 is a novel tardigrade-specific gene conferring resistance to IR. Our study sheds light on mechanisms of DNA repair helping cope with high levels of DNA damage inflicted by IR.
Asunto(s)
Reparación del ADN , Proteínas de Unión al ADN , Radiación Ionizante , Tardigrada , Transcriptoma , Tardigrada/genética , Tardigrada/metabolismo , Animales , Humanos , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Perfilación de la Expresión Génica , Daño del ADN , Tolerancia a Radiación/genéticaRESUMEN
Urp1 and Urp2 are two neuropeptides, members of the Urotensin 2 family, that have been recently involved in the control of body axis morphogenesis in zebrafish. They are produced by a population of sensory spinal neurons, called cerebrospinal fluid contacting neurons (CSF-cNs), under the control of signals relying on the Reissner fiber, an extracellular thread bathing in the CSF. Here, we have investigated further the function of Urp1 and Urp2 (Urp1/2) in body axis formation and maintenance. We showed that urp1;urp2 double mutants develop strong body axis defects during larval growth, revealing the redundancy between the two neuropeptides. These defects were similar to those previously reported in uts2r3 mutants. We observed that this phenotype is not associated with congenital defects in vertebrae formation, but by using specific inhibitors, we found that, at least in the embryo, the action of Urp1/2 signaling depends on myosin II contraction. Finally, we provide evidence that while the Urp1/2 signaling is functioning during larval growth, it is dispensable for embryonic development. Taken together, our results show that Urp1/2 signaling is required in larvae to promote correct vertebral body axis, most likely by regulating muscle tone.
Asunto(s)
Neuropéptidos , Pez Cebra , Animales , Larva , Columna Vertebral , Morfogénesis , Proteínas de Pez CebraRESUMEN
BACKGROUND: Anaplastic large cell lymphoma positive for ALK (ALK+ ALCL) is a rare type of non-Hodgkin lymphoma. This lymphoma is caused by chromosomal translocations involving the anaplastic lymphoma kinase gene (ALK). In this study, we aimed to identify mechanisms of transformation and therapeutic targets by generating a model of ALK+ ALCL lymphomagenesis ab initio with the specific NPM-ALK fusion. METHODS: We performed CRISPR/Cas9-mediated genome editing of the NPM-ALK chromosomal translocation in primary human activated T lymphocytes. RESULTS: Both CD4+ and CD8+ NPM-ALK-edited T lymphocytes showed rapid and reproducible competitive advantage in culture and led to in vivo disease development with nodal and extra-nodal features. Murine tumors displayed the phenotypic diversity observed in ALK+ ALCL patients, including CD4+ and CD8+ lymphomas. Assessment of transcriptome data from models and patients revealed global activation of the WNT signaling pathway, including both canonical and non-canonical pathways, during ALK+ ALCL lymphomagenesis. Specifically, we found that the WNT signaling cell surface receptor ROR2 represented a robust and genuine marker of all ALK+ ALCL patient tumor samples. CONCLUSIONS: In this study, ab initio modeling of the ALK+ ALCL chromosomal translocation in mature T lymphocytes enabled the identification of new therapeutic targets. As ROR2 targeting approaches for other cancers are under development (including lung and ovarian tumors), our findings suggest that ALK+ ALCL cases with resistance to current therapies may also benefit from ROR2 targeting strategies.
Asunto(s)
Linfoma Anaplásico de Células Grandes , Quinasa de Linfoma Anaplásico/genética , Animales , Humanos , Linfoma Anaplásico de Células Grandes/genética , Linfoma Anaplásico de Células Grandes/metabolismo , Linfoma Anaplásico de Células Grandes/patología , Ratones , Fenotipo , Proteínas Tirosina Quinasas/metabolismo , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/genética , Translocación GenéticaRESUMEN
Sickle cell disease (SCD) is caused by a mutation in the ß-globin gene leading to polymerization of the sickle hemoglobin (HbS) and deformation of red blood cells. Autologous transplantation of hematopoietic stem/progenitor cells (HSPCs) genetically modified using lentiviral vectors (LVs) to express an anti-sickling ß-globin leads to some clinical benefit in SCD patients, but it requires high-level transgene expression (i.e., high vector copy number [VCN]) to counteract HbS polymerization. Here, we developed therapeutic approaches combining LV-based gene addition and CRISPR-Cas9 strategies aimed to either knock down the sickle ß-globin and increase the incorporation of an anti-sickling globin (AS3) in hemoglobin tetramers, or to induce the expression of anti-sickling fetal γ-globins. HSPCs from SCD patients were transduced with LVs expressing AS3 and a guide RNA either targeting the endogenous ß-globin gene or regions involved in fetal hemoglobin silencing. Transfection of transduced cells with Cas9 protein resulted in high editing efficiency, elevated levels of anti-sickling hemoglobins, and rescue of the SCD phenotype at a significantly lower VCN compared to the conventional LV-based approach. This versatile platform can improve the efficacy of current gene addition approaches by combining different therapeutic strategies, thus reducing the vector amount required to achieve a therapeutic VCN and the associated genotoxicity risk.
Asunto(s)
Anemia de Células Falciformes , Edición Génica , Anemia de Células Falciformes/genética , Anemia de Células Falciformes/terapia , Proteína 9 Asociada a CRISPR/genética , Hemoglobina Fetal/genética , Edición Génica/métodos , Humanos , Globinas beta/genéticaRESUMEN
Ewing sarcoma is characterized by pathognomonic translocations, most frequently fusing EWSR1 with FLI1. An estimated 30% of Ewing sarcoma tumors also display genetic alterations in STAG2, TP53, or CDKN2A (SPC). Numerous attempts to develop relevant Ewing sarcoma models from primary human cells have been unsuccessful in faithfully recapitulating the phenotypic, transcriptomic, and epigenetic features of Ewing sarcoma. In this study, by engineering the t(11;22)(q24;q12) translocation together with a combination of SPC mutations, we generated a wide collection of immortalized cells (EWIma cells) tolerating EWSR1-FLI1 expression from primary mesenchymal stem cells (MSC) derived from a patient with Ewing sarcoma. Within this model, SPC alterations strongly favored Ewing sarcoma oncogenicity. Xenograft experiments with independent EWIma cells induced tumors and metastases in mice, which displayed bona fide features of Ewing sarcoma. EWIma cells presented balanced but also more complex translocation profiles mimicking chromoplexy, which is frequently observed in Ewing sarcoma and other cancers. Collectively, these results demonstrate that bone marrow-derived MSCs are a source of origin for Ewing sarcoma and also provide original experimental models to investigate Ewing sarcomagenesis. SIGNIFICANCE: These findings demonstrate that Ewing sarcoma can originate from human bone-marrow-derived mesenchymal stem cells and that recurrent mutations support EWSR1-FLI1 translocation-mediated transformation.
Asunto(s)
Transformación Celular Neoplásica , Susceptibilidad a Enfermedades , Células Madre Mesenquimatosas/metabolismo , Sarcoma de Ewing/etiología , Sarcoma de Ewing/metabolismo , Animales , Biomarcadores , Sistemas CRISPR-Cas , Células Cultivadas , Biología Computacional/métodos , Modelos Animales de Enfermedad , Edición Génica , Perfilación de la Expresión Génica , Reordenamiento Génico , Marcación de Gen , Xenoinjertos , Humanos , Inmunofenotipificación , Hibridación Fluorescente in Situ , Células Madre Mesenquimatosas/patología , Ratones , Mutación , Sarcoma de Ewing/patología , Translocación GenéticaRESUMEN
Sickle cell disease (SCD) is caused by a single amino acid change in the adult hemoglobin (Hb) ß chain that causes Hb polymerization and red blood cell (RBC) sickling. The co-inheritance of mutations causing fetal γ-globin production in adult life hereditary persistence of fetal Hb (HPFH) reduces the clinical severity of SCD. HPFH mutations in the HBG γ-globin promoters disrupt binding sites for the repressors BCL11A and LRF. We used CRISPR-Cas9 to mimic HPFH mutations in the HBG promoters by generating insertions and deletions, leading to disruption of known and putative repressor binding sites. Editing of the LRF-binding site in patient-derived hematopoietic stem/progenitor cells (HSPCs) resulted in γ-globin derepression and correction of the sickling phenotype. Xenotransplantation of HSPCs treated with gRNAs targeting the LRF-binding site showed a high editing efficiency in repopulating HSPCs. This study identifies the LRF-binding site as a potent target for genome-editing treatment of SCD.
Asunto(s)
Anemia de Células Falciformes , Talasemia beta , Anemia de Células Falciformes/genética , Anemia de Células Falciformes/terapia , Sitios de Unión , Sistemas CRISPR-Cas , Hemoglobina Fetal/genética , Hemoglobina Fetal/metabolismo , Edición Génica/métodos , Humanos , Fenotipo , Globinas beta/genética , Globinas beta/metabolismo , Talasemia beta/genética , Talasemia beta/metabolismo , Talasemia beta/terapia , gamma-Globinas/genética , gamma-Globinas/metabolismoRESUMEN
Sex pheromone receptors (PRs) are key players in chemical communication between mating partners in insects. In the highly diversified insect order Lepidoptera, male PRs tuned to female-emitted type I pheromones (which make up the vast majority of pheromones identified) form a dedicated subfamily of odorant receptors (ORs). Here, using a combination of heterologous expression and in vivo genome editing methods, we bring functional evidence that at least one moth PR does not belong to this subfamily but to a distantly related OR lineage. This PR, identified in the cotton leafworm Spodoptera littoralis, is highly expressed in male antennae and is specifically tuned to the major sex pheromone component emitted by females. Together with a comprehensive phylogenetic analysis of moth ORs, our functional data suggest two independent apparitions of PRs tuned to type I pheromones in Lepidoptera, opening up a new path for studying the evolution of moth pheromone communication.
Asunto(s)
Proteínas de Insectos/metabolismo , Mariposas Nocturnas/metabolismo , Receptores de Feromonas/metabolismo , Atractivos Sexuales/metabolismo , Animales , Conducta Animal , Sistemas CRISPR-Cas , Drosophila/genética , Drosophila/metabolismo , Femenino , Regulación de la Expresión Génica , Técnicas de Inactivación de Genes , Proteínas de Insectos/genética , Lepidópteros/genética , Lepidópteros/metabolismo , Masculino , Mariposas Nocturnas/genética , Receptores Odorantes , Receptores de Feromonas/clasificación , Receptores de Feromonas/genética , Spodoptera/genética , Spodoptera/metabolismo , Transcriptoma , Xenopus/genética , Xenopus/metabolismoRESUMEN
Targeted mutagenesis using CRISPR/Cas9 technology has been shown to be a powerful approach to examine gene function in diverse metazoan species. One common drawback is that mixed genotypes, and thus variable phenotypes, arise in the F0 generation because incorrect DNA repair produces different mutations amongst cells of the developing embryo. We report here an effective method for gene knockout (KO) in the hydrozoan Clytia hemisphaerica, by injection into the egg of Cas9/sgRNA ribonucleoprotein complex (RNP). Expected phenotypes were observed in the F0 generation when targeting endogenous GFP genes, which abolished fluorescence in embryos, or CheRfx123 (that codes for a conserved master transcriptional regulator for ciliogenesis) which caused sperm motility defects. When high concentrations of Cas9 RNP were used, the mutations in target genes at F0 polyp or jellyfish stages were not random but consisted predominantly of one or two specific deletions between pairs of short microhomologies flanking the cleavage site. Such microhomology-mediated (MM) deletion is most likely caused by microhomology-mediated end-joining (MMEJ), which may be favoured in early stage embryos. This finding makes it very easy to isolate uniform, largely non-mosaic mutants with predictable genotypes in the F0 generation in Clytia, allowing rapid and reliable phenotype assessment.
Asunto(s)
Sistemas CRISPR-Cas/genética , Ribonucleoproteínas/metabolismo , Animales , Femenino , Técnicas de Inactivación de Genes/métodos , Hidrozoos/genética , Hidrozoos/metabolismo , Masculino , Mosaicismo , Ribonucleoproteínas/genéticaRESUMEN
Cis-regulation plays an essential role in the control of gene expression, and is particularly complex and poorly understood for developmental genes, which are subject to multiple levels of modulation. In this study, we performed a global analysis of the cis-acting elements involved in the control of the zebrafish developmental gene krox20. krox20 encodes a transcription factor required for hindbrain segmentation and patterning, a morphogenetic process highly conserved during vertebrate evolution. Chromatin accessibility analysis reveals a cis-regulatory landscape that includes 6 elements participating in the control of initiation and autoregulatory aspects of krox20 hindbrain expression. Combining transgenic reporter analyses and CRISPR/Cas9-mediated mutagenesis, we assign precise functions to each of these 6 elements and provide a comprehensive view of krox20 cis-regulation. Three important features emerged. First, cooperation between multiple cis-elements plays a major role in the regulation. Cooperation can surprisingly combine synergy and redundancy, and is not restricted to transcriptional enhancer activity (for example, 4 distinct elements cooperate through different modes to maintain autoregulation). Second, several elements are unexpectedly versatile, which allows them to be involved in different aspects of control of gene expression. Third, comparative analysis of the elements and their activities in several vertebrate species reveals that this versatility is underlain by major plasticity across evolution, despite the high conservation of the gene expression pattern. These characteristics are likely to be of broad significance for developmental genes.
Asunto(s)
Proteína 2 de la Respuesta de Crecimiento Precoz/genética , Regulación del Desarrollo de la Expresión Génica , Rombencéfalo/metabolismo , Proteínas de Pez Cebra/genética , Pez Cebra/genética , Secuencia de Aminoácidos , Animales , Sistemas CRISPR-Cas , Cromatina/metabolismo , Proteína 2 de la Respuesta de Crecimiento Precoz/fisiología , Elementos de Facilitación Genéticos , Evolución Molecular , Sitios Genéticos , Morfogénesis/genética , Activación Transcripcional , Pez Cebra/embriologíaRESUMEN
The generation of gene-edited animals using the CRISPRs/Cas9 system is based on microinjection into zygotes which is inefficient, time consuming and demands high technical skills. We report the optimization of an electroporation method for intact rat zygotes using sgRNAs and Cas9 protein in combination or not with ssODNs (~100 nt). This resulted in high frequency of knockouts, between 15 and 50% of analyzed animals. Importantly, using ssODNs as donor template resulted in precise knock-in mutations in 25-100% of analyzed animals, comparable to microinjection. Electroporation of long ssDNA or dsDNA donors successfully used in microinjection in the past did not allow generation of genome-edited animals despite dsDNA visualization within zygotes. Thus, simultaneous electroporation of a large number of intact rat zygotes is a rapid, simple, and efficient method for the generation of a variety of genome-edited rats.
Asunto(s)
Proteína 9 Asociada a CRISPR/metabolismo , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/fisiología , Cigoto/metabolismo , Animales , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Electroporación/métodos , Femenino , Genotipo , Microscopía Confocal , Mutación , RatasRESUMEN
Lepidoptera suffer critical lack of genetic tools and heritable genome edition has been achieved only in a few model species. Here we demonstrate that the CRISPR/Cas9 system is highly efficient for genome editing in a non-model crop pest Lepidoptera, the noctuid moth Spodoptera littoralis. We knocked-out the olfactory receptor co-receptor Orco gene to investigate its function in Lepidoptera olfaction. We find that 89.6% of the injected individuals carried Orco mutations, 70% of which transmitted them to the next generation. CRISPR/Cas9-mediated Orco knockout caused defects in plant odor and sex pheromone olfactory detection in homozygous individuals. Our work genetically defines Orco as an essential OR partner for both host and mate detection in Lepidoptera, and demonstrates that CRISPR/Cas9 is a simple and highly efficient genome editing technique in noctuid pests opening new routes for gene function analysis and the development of novel pest control strategies.
Asunto(s)
Sistemas CRISPR-Cas , Edición Génica/métodos , Trastornos del Olfato/genética , Spodoptera/genética , Animales , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Proteínas de Insectos/genética , Mutación , Receptores Odorantes/genética , Atractivos Sexuales/genética , Spodoptera/fisiologíaRESUMEN
Genome editing has now been reported in many systems using TALEN and CRISPR-Cas9 nucleases. Precise mutations can be introduced during homology-directed repair with donor DNA carrying the wanted sequence edit, but efficiency is usually lower than for gene knockout and optimal strategies have not been extensively investigated. Here, we show that using phosphorothioate-modified oligonucleotides strongly enhances genome editing efficiency of single-stranded oligonucleotide donors in cultured cells. In addition, it provides better design flexibility, allowing insertions more than 100 bp long. Despite previous reports of phosphorothioate-modified oligonucleotide toxicity, clones of edited cells are readily isolated and targeted sequence insertions are achieved in rats and mice with very high frequency, allowing for homozygous loxP site insertion at the mouse ROSA locus in particular. Finally, when detected, imprecise knockin events exhibit indels that are asymmetrically positioned, consistent with genome editing taking place by two steps of single-strand annealing.
Asunto(s)
Sistemas CRISPR-Cas , Endonucleasas/genética , Edición Génica , Nucleasas de los Efectores Tipo Activadores de la Transcripción/genética , Animales , Secuencia de Bases , Línea Celular Tumoral , Marcación de Gen , Humanos , Mutación INDEL , Ratones , Oligonucleótidos/genética , Ratas , Pez CebraRESUMEN
The rat is an important animal model to understand gene function and model human diseases. Since recent years, the development of gene-specific nucleases has become important for generating new rat models of human diseases, to analyze the role of genes and to generate human antibodies. Transcription activator-like (TALE) nucleases efficiently create gene-specific knockout rats and lead to the possibility of gene targeting by homology-directed recombination (HDR) and generating knock-in rats. We describe a detailed protocol for generating knockout and knock-in rats via microinjection of TALE nucleases into fertilized eggs. This technology is an efficient, cost- and time-effective method for creating new rat models.
Asunto(s)
Animales Modificados Genéticamente/genética , Endonucleasas/genética , Técnicas de Inactivación de Genes/métodos , Animales , Genoma , Recombinación Homóloga/genética , Humanos , ARN Mensajero/genética , Ratas , Transactivadores/genéticaRESUMEN
The generation of genetically-modified organisms has been revolutionized by the development of new genome editing technologies based on the use of gene-specific nucleases, such as meganucleases, ZFNs, TALENs and CRISPRs-Cas9 systems. The most rapid and cost-effective way to generate genetically-modified animals is by microinjection of the nucleic acids encoding gene-specific nucleases into zygotes. However, the efficiency of the procedure can still be improved. In this work we aim to increase the efficiency of CRISPRs-Cas9 and TALENs homology-directed repair by using TALENs and Cas9 proteins, instead of mRNA, microinjected into rat and mouse zygotes along with long or short donor DNAs. We observed that Cas9 protein was more efficient at homology-directed repair than mRNA, while TALEN protein was less efficient than mRNA at inducing homology-directed repair. Our results indicate that the use of Cas9 protein could represent a simple and practical methodological alternative to Cas9 mRNA in the generation of genetically-modified rats and mice as well as probably some other mammals.
Asunto(s)
Sistemas CRISPR-Cas/genética , Ingeniería de Proteínas , Reparación del ADN por Recombinación/genética , Cigoto/fisiología , Animales , Ratones , Ratones Endogámicos C57BL , Microinyecciones , Ratas , Ratas Sprague-DawleyRESUMEN
A few animal models of Duchenne muscular dystrophy (DMD) are available, large ones such as pigs or dogs being expensive and difficult to handle. Mdx (X-linked muscular dystrophy) mice only partially mimic the human disease, with limited chronic muscular lesions and muscle weakness. Their small size also imposes limitations on analyses. A rat model could represent a useful alternative since rats are small animals but 10 times bigger than mice and could better reflect the lesions and functional abnormalities observed in DMD patients. Two lines of Dmd mutated-rats (Dmdmdx) were generated using TALENs targeting exon 23. Muscles of animals of both lines showed undetectable levels of dystrophin by western blot and less than 5% of dystrophin positive fibers by immunohistochemistry. At 3 months, limb and diaphragm muscles from Dmdmdx rats displayed severe necrosis and regeneration. At 7 months, these muscles also showed severe fibrosis and some adipose tissue infiltration. Dmdmdx rats showed significant reduction in muscle strength and a decrease in spontaneous motor activity. Furthermore, heart morphology was indicative of dilated cardiomyopathy associated histologically with necrotic and fibrotic changes. Echocardiography showed significant concentric remodeling and alteration of diastolic function. In conclusion, Dmdmdx rats represent a new faithful small animal model of DMD.
Asunto(s)
Modelos Animales de Enfermedad , Distrofina/deficiencia , Distrofia Muscular Animal , Distrofia Muscular de Duchenne/genética , Animales , Secuencia de Bases , Creatina Quinasa/sangre , Distrofina/genética , Distrofina/metabolismo , Exones , Femenino , Fibrosis , Eliminación de Gen , Expresión Génica , Marcación de Gen , Masculino , Debilidad Muscular/genética , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/patología , Distrofia Muscular de Duchenne/fisiopatología , Mutación , Miocardio/metabolismo , Miocardio/patología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Remodelación Ventricular/genéticaRESUMEN
The generation of genetically modified animals is important for both research and commercial purposes. The rat is an important model organism that until recently lacked efficient genetic engineering tools. Sequence-specific nucleases, such as ZFNs, TALE nucleases, and CRISPR/Cas9 have allowed the creation of rat knockout models. Genetic engineering by homology-directed repair (HDR) is utilized to create animals expressing transgenes in a controlled way and to introduce precise genetic modifications. We applied TALE nucleases and donor DNA microinjection into zygotes to generate HDR-modified rats with large new sequences introduced into three different loci with high efficiency (0.62%-5.13% of microinjected zygotes). Two of these loci (Rosa26 and Hprt1) are known to allow robust and reproducible transgene expression and were targeted for integration of a GFP expression cassette driven by the CAG promoter. GFP-expressing embryos and four Rosa26 GFP rat lines analyzed showed strong and widespread GFP expression in most cells of all analyzed tissues. The third targeted locus was Ighm, where we performed successful exon exchange of rat exon 2 for the human one. At all three loci we observed HDR only when using linear and not circular donor DNA. Mild hypothermic (30°C) culture of zygotes after microinjection increased HDR efficiency for some loci. Our study demonstrates that TALE nuclease and donor DNA microinjection into rat zygotes results in efficient and reproducible targeted donor integration by HDR. This allowed creation of genetically modified rats in a work-, cost-, and time-effective manner.
Asunto(s)
Marcación de Gen , Ingeniería Genética , Animales , Secuencia de Bases , Células Cultivadas , Enzimas de Restricción del ADN/biosíntesis , Enzimas de Restricción del ADN/genética , Femenino , Hipoxantina Fosforribosiltransferasa/genética , Masculino , Microinyecciones , Ratas Sprague-Dawley , Ratas Transgénicas , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Reparación del ADN por Recombinación , CigotoRESUMEN
The rat is a model of choice to understanding gene function and modeling human diseases. Since recent years, successful engineering technologies using gene-specific nucleases have been developed to gene edit the genome of different species, including the rat. This development has become important for the creation of new rat animals models of human diseases, analyze the role of genes and express recombinant proteins. Transcription activator-like (TALE) nucleases are designed nucleases consist of a DNA binding domain fused to a nuclease domain capable of cleaving the targeted DNA. We describe a detailed protocol for generating knockout rats via microinjection of TALE nucleases into fertilized eggs. This technology is an efficient, cost- and time-effective method for creating new rat models.
Asunto(s)
Técnicas de Inactivación de Genes , Mutagénesis Sitio-Dirigida/métodos , Animales , Reparación del ADN por Unión de Extremidades , Desoxirribonucleasas/química , Desoxirribonucleasas/genética , Transferencia de Embrión , Embrión de Mamíferos , Femenino , Recombinación Homóloga , Microinyecciones , Ratas , Ratas Sprague-DawleyRESUMEN
Sequence-specific nucleases like TALENs and the CRISPR/Cas9 system have greatly expanded the genome editing possibilities in model organisms such as zebrafish. Both systems have recently been used to create knock-out alleles with great efficiency, and TALENs have also been successfully employed in knock-in of DNA cassettes at defined loci via homologous recombination (HR). Here we report CRISPR/Cas9-mediated knock-in of DNA cassettes into the zebrafish genome at a very high rate by homology-independent double-strand break (DSB) repair pathways. After co-injection of a donor plasmid with a short guide RNA (sgRNA) and Cas9 nuclease mRNA, concurrent cleavage of donor plasmid DNA and the selected chromosomal integration site resulted in efficient targeted integration of donor DNA. We successfully employed this approach to convert eGFP into Gal4 transgenic lines, and the same plasmids and sgRNAs can be applied in any species where eGFP lines were generated as part of enhancer and gene trap screens. In addition, we show the possibility of easily targeting DNA integration at endogenous loci, thus greatly facilitating the creation of reporter and loss-of-function alleles. Due to its simplicity, flexibility, and very high efficiency, our method greatly expands the repertoire for genome editing in zebrafish and can be readily adapted to many other organisms.
Asunto(s)
Proteínas Asociadas a CRISPR/metabolismo , Reparación del ADN , Técnicas de Sustitución del Gen , Ingeniería Genética/métodos , Pez Cebra/genética , Animales , Proteínas Asociadas a CRISPR/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Roturas del ADN de Doble Cadena , Genoma , Mutagénesis , Reparación del ADN por Recombinación , Pez Cebra/embriología , ARN Pequeño no TraducidoRESUMEN
Chromosomal translocations are signatures of numerous cancers and lead to expression of fusion genes that act as oncogenes. The wealth of genomic aberrations found in cancer, however, makes it challenging to assign a specific phenotypic change to a specific aberration. In this study, we set out to use genome editing with zinc finger (ZFN) and transcription activator-like effector (TALEN) nucleases to engineer, de novo, translocation-associated oncogenes at cognate endogenous loci in human cells. Using ZFNs and TALENs designed to cut precisely at relevant translocation breakpoints, we induced cancer-relevant t(11;22)(q24;q12) and t(2;5)(p23;q35) translocations found in Ewing sarcoma and anaplastic large cell lymphoma (ALCL), respectively. We recovered both translocations with high efficiency, resulting in the expression of the EWSR1-FLI1 and NPM1-ALK fusions. Breakpoint junctions recovered after ZFN cleavage in human embryonic stem (ES) cell-derived mesenchymal precursor cells fully recapitulated the genomic characteristics found in tumor cells from Ewing sarcoma patients. This approach with tailored nucleases demonstrates that expression of fusion genes found in cancer cells can be induced from the native promoter, allowing interrogation of both the underlying mechanisms and oncogenic consequences of tumor-related translocations in human cells. With an analogous strategy, the ALCL translocation was reverted in a patient cell line to restore the integrity of the two participating chromosomes, further expanding the repertoire of genomic rearrangements that can be engineered by tailored nucleases.
Asunto(s)
Endonucleasas/metabolismo , Neoplasias/enzimología , Neoplasias/genética , Translocación Genética , Dedos de Zinc , Línea Celular , Puntos de Rotura del Cromosoma , Humanos , Nucleofosmina , Proteínas Tirosina Quinasas/genética , Sarcoma de Ewing/genética , Sarcoma de Ewing/metabolismoRESUMEN
Guanine quadruplexes (G4) are unusual four-stranded nucleic acid structures formed by G-rich DNA/RNA. Beyond their likely biological relevance, the self-assembly, stability, and rigidity of these structures are also interesting for nanotechnology and biotechnology applications. Therefore, efforts are carried out to understand the rules that govern stability and folding of G-quadruplexes. We focus this chapter on tetramolecular conformations which are simple tractable models. We report here the experimental parameters, molecules, and modifications that affect thermal stability and/or association kinetics of these structures. Some chemical modifications which facilitate tetramolecular quadruplex formation and can be useful for nano- or biotechnology are also described.