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1.
Elife ; 132024 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-38980300

RESUMO

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.


Assuntos
Reparo do DNA , Proteínas de Ligação a DNA , Radiação Ionizante , Tardígrados , Transcriptoma , Tardígrados/genética , Tardígrados/metabolismo , Animais , Humanos , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Perfilação da Expressão Gênica , Dano ao DNA , Tolerância a Radiação/genética
2.
Nat Commun ; 13(1): 6618, 2022 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-36333351

RESUMO

Sickle cell disease and ß-thalassemia affect the production of the adult ß-hemoglobin chain. The clinical severity is lessened by mutations that cause fetal γ-globin expression in adult life (i.e., the hereditary persistence of fetal hemoglobin). Mutations clustering ~200 nucleotides upstream of the HBG transcriptional start sites either reduce binding of the LRF repressor or recruit the KLF1 activator. Here, we use base editing to generate a variety of mutations in the -200 region of the HBG promoters, including potent combinations of four to eight γ-globin-inducing mutations. Editing of patient hematopoietic stem/progenitor cells is safe, leads to fetal hemoglobin reactivation and rescues the pathological phenotype. Creation of a KLF1 activator binding site is the most potent strategy - even in long-term repopulating hematopoietic stem/progenitor cells. Compared with a Cas9-nuclease approach, base editing avoids the generation of insertions, deletions and large genomic rearrangements and results in higher γ-globin levels. Our results demonstrate that base editing of HBG promoters is a safe, universal strategy for treating ß-hemoglobinopathies.


Assuntos
Anemia Falciforme , Talassemia beta , Humanos , Hemoglobina Fetal/genética , Hemoglobina Fetal/metabolismo , gama-Globinas/genética , Talassemia beta/genética , Talassemia beta/terapia , Anemia Falciforme/genética , Células-Tronco Hematopoéticas/metabolismo
3.
Nat Commun ; 13(1): 3435, 2022 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-35701478

RESUMO

Base Editors are emerging as an innovative technology to introduce point mutations in complex genomes. So far, the requirement of an NGG Protospacer Adjacent Motif (PAM) at a suitable position often limits the base editing possibility to model human pathological mutations in animals. Here we show that, using the CBE4max-SpRY variant recognizing nearly all PAM sequences, we could introduce point mutations for the first time in an animal model with high efficiency, thus drastically increasing the base editing possibilities. With this near PAM-less base editor we could simultaneously mutate several genes and we developed a co-selection method to identify the most edited embryos based on a simple visual screening. Finally, we apply our method to create a zebrafish model for melanoma predisposition based on the simultaneous base editing of multiple genes. Altogether, our results considerably expand the Base Editor application to introduce human disease-causing mutations in zebrafish.


Assuntos
Proteína 9 Associada à CRISPR , Edição de Genes , Animais , Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Genoma/genética , Peixe-Zebra/genética , Peixe-Zebra/metabolismo
4.
Mol Cancer ; 21(1): 65, 2022 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-35246138

RESUMO

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.


Assuntos
Linfoma Anaplásico de Células Grandes , Quinase do Linfoma Anaplásico/genética , Animais , Humanos , Linfoma Anaplásico de Células Grandes/genética , Linfoma Anaplásico de Células Grandes/metabolismo , Linfoma Anaplásico de Células Grandes/patologia , Camundongos , Fenótipo , Proteínas Tirosina Quinases/metabolismo , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/genética , Translocação Genética
5.
Hum Gene Ther ; 32(19-20): 1059-1075, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34494480

RESUMO

Recent advances in genome editing tools, especially novel developments in the clustered regularly interspaced short palindromic repeats associated to Cas9 nucleases (CRISPR/Cas9)-derived editing machinery, have revolutionized not only basic science but, importantly, also the gene therapy field. Their flexibility and ability to introduce precise modifications in the genome to disrupt or correct genes or insert expression cassettes in safe harbors in the genome underline their potential applications as a medicine of the future to cure many genetic diseases. In this review, we give an overview of the recent progress made by French researchers in the field of therapeutic genome editing, while putting their work in the general context of advances made in the field. We focus on recent hematopoietic stem cell gene editing strategies for blood diseases affecting the red blood cells or blood coagulation as well as lysosomal storage diseases. We report on a genome editing-based therapy for muscular dystrophy and the potency of T cell gene editing to increase anticancer activity of chimeric antigen receptor T cells to combat cancer. We will also discuss technical obstacles and side effects such as unwanted editing activity that need to be surmounted on the way toward a clinical implementation of genome editing. We propose here improvements developed today, including by French researchers to overcome the editing-related genotoxicity and improve editing precision by the use of novel recombinant nuclease-based systems such as nickases, base editors, and prime editors. Finally, a solution is proposed to resolve the cellular toxicity induced by the systems employed for gene editing machinery delivery.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Sistemas CRISPR-Cas/genética , Endonucleases/genética , Técnicas de Transferência de Genes , Terapia Genética
6.
Cancer Res ; 81(19): 4994-5006, 2021 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-34341072

RESUMO

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.


Assuntos
Transformação Celular Neoplásica , Suscetibilidade a Doenças , Células-Tronco Mesenquimais/metabolismo , Sarcoma de Ewing/etiologia , Sarcoma de Ewing/metabolismo , Animais , Biomarcadores , Sistemas CRISPR-Cas , Células Cultivadas , Biologia Computacional/métodos , Modelos Animais de Doenças , Edição de Genes , Perfilação da Expressão Gênica , Rearranjo Gênico , Marcação de Genes , Xenoenxertos , Humanos , Imunofenotipagem , Hibridização in Situ Fluorescente , Células-Tronco Mesenquimais/patologia , Camundongos , Mutação , Sarcoma de Ewing/patologia , Translocação Genética
7.
Sci Adv ; 6(7)2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32917636

RESUMO

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.


Assuntos
Anemia Falciforme , Talassemia beta , Anemia Falciforme/genética , Anemia Falciforme/terapia , Sítios de Ligação , Sistemas CRISPR-Cas , Hemoglobina Fetal/genética , Hemoglobina Fetal/metabolismo , Edição de Genes/métodos , Humanos , Fenótipo , Globinas beta/genética , Globinas beta/metabolismo , Talassemia beta/genética , Talassemia beta/metabolismo , Talassemia beta/terapia , gama-Globinas/genética , gama-Globinas/metabolismo
8.
PLoS Genet ; 15(10): e1008355, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31584931

RESUMO

Deficiency in several of the classical human RAD51 paralogs [RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3] is associated with cancer predisposition and Fanconi anemia. To investigate their functions, isogenic disruption mutants for each were generated in non-transformed MCF10A mammary epithelial cells and in transformed U2OS and HEK293 cells. In U2OS and HEK293 cells, viable ablated clones were readily isolated for each RAD51 paralog; in contrast, with the exception of RAD51B, RAD51 paralogs are cell-essential in MCF10A cells. Underlining their importance for genomic stability, mutant cell lines display variable growth defects, impaired sister chromatid recombination, reduced levels of stable RAD51 nuclear foci, and hyper-sensitivity to mitomycin C and olaparib, with the weakest phenotypes observed in RAD51B-deficient cells. Altogether these observations underscore the contributions of RAD51 paralogs in diverse DNA repair processes, and demonstrate essential differences in different cell types. Finally, this study will provide useful reagents to analyze patient-derived mutations and to investigate mechanisms of chemotherapeutic resistance deployed by cancers.


Assuntos
Reparo do DNA/genética , Proteínas de Ligação a DNA/genética , Recombinação Homóloga/genética , Rad51 Recombinase/genética , Núcleo Celular/genética , Cromátides/genética , Dano ao DNA/genética , Genoma Humano/genética , Células HEK293 , Humanos , Mutação
9.
iScience ; 5: 19-29, 2018 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-30240643

RESUMO

Circular RNAs constitute a unique class of RNAs whose precise functions remain to be elucidated. In particular, cancer-associated chromosomal translocations can give rise to fusion circular RNAs that play a role in leukemia progression. However, how and when fusion circular RNAs are formed and whether they are being selected in cancer cells remains unknown. Here, we used CRISPR/Cas9 to generate physiological translocation models of NPM1-ALK fusion gene. We showed that, in addition to generating fusion proteins and activating specific oncogenic pathways, chromosomal translocation induced by CRISPR/Cas9 led to the formation of de novo fusion circular RNAs. Specifically, we could recover different classes of circular RNAs composed of different circularization junctions, mainly back-spliced species. In addition, we identified fusion circular RNAs identical to those found in related patient tumor cells providing evidence that fusion circular RNAs arise early after chromosomal formation and are not just a consequence of the oncogenesis process.

10.
Sci Rep ; 8(1): 11734, 2018 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-30082705

RESUMO

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.


Assuntos
Sistemas CRISPR-Cas/genética , Ribonucleoproteínas/metabolismo , Animais , Feminino , Técnicas de Inativação de Genes/métodos , Hidrozoários/genética , Hidrozoários/metabolismo , Masculino , Mosaicismo , Ribonucleoproteínas/genética
11.
Transplantation ; 102(8): 1271-1278, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29688994

RESUMO

BACKGROUND: Immunodeficient mice are invaluable tools to analyze the long-term effects of potentially immunogenic molecules in the absence of adaptive immune responses. Nevertheless, there are models and experimental situations that would beneficiate of larger immunodeficient recipients. Rats are ideally suited to perform experiments in which larger size is needed and are still a small animal model suitable for rodent facilities. Additionally, rats reproduce certain human diseases better than mice, such as ankylosing spondylitis and Duchenne disease, and these disease models would greatly benefit from immunodeficient rats to test different immunogenic treatments. METHODS: We describe the generation of Il2rg-deficient rats and their crossing with previously described Rag1-deficient rats to generate double-mutant RRG animals. RESULTS: As compared with Rag1-deficient rats, Il2rg-deficient rats were more immunodeficient because they partially lacked not only T and B cells but also NK cells. RRG animals showed a more profound immunossuppressed phenotype because they displayed undetectable levels of T, B, and NK cells. Similarly, all immunoglobulin isotypes in sera were decreased in Rag1- or Il2rg-deficient rats and undetectable in Rats Rag1 and Il2rg (RRG) animals. Rag1- or Il2rg-deficient rats rejected allogeneic skin transplants and human tumors, whereas animals not only accepted allogeneic rat skin but also xenogeneic human tumors, skin, and hepatocytes. Immune humanization of RRG animals was unsuccessful. CONCLUSIONS: Thus, immunodeficient RRG animals are useful recipients for long-term studies in which immune responses could be an obstacle, including tissue humanization of different tissues.


Assuntos
Deleção de Genes , Proteínas de Homeodomínio/genética , Subunidade gama Comum de Receptores de Interleucina/genética , Animais , Animais Geneticamente Modificados , Cruzamentos Genéticos , Modelos Animais de Doenças , Éxons , Feminino , Genótipo , Hepatócitos/citologia , Humanos , Sistema Imunitário , Fígado/imunologia , Masculino , Mutação , Ratos , Ratos Sprague-Dawley , Transplante de Pele , Transplante Heterólogo , Transplantes
12.
Mult Scler ; 24(11): 1421-1432, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-28752787

RESUMO

BACKGROUND: In multiple sclerosis, development of screening tools for remyelination-promoting molecules is timely. OBJECTIVE: A Xenopus transgenic line allowing conditional ablation of myelinating oligodendrocytes has been adapted for in vivo screening of remyelination-favoring molecules. METHODS: In this transgenic, the green fluorescent protein reporter is fused to E. coli nitroreductase and expressed specifically in myelinating oligodendrocytes. Nitroreductase converts the innocuous pro-drug metronidazole to a cytotoxin. Spontaneous remyelination occurs after metronidazole-induced demyelinating responses. As tadpoles are transparent, these events can be monitored in vivo and quantified. At the end of metronidazole-induced demyelination, tadpoles were screened in water containing the compounds tested. After 72 h, remyelination was assayed by counting numbers of oligodendrocytes per optic nerve. RESULTS: Among a battery of molecules tested, siponimod, a dual agonist of sphingosine-1-phosphate receptor 1 and 5, was among the most efficient favoring remyelination. Crispr/cas9 gene editing showed that the promyelinating effect of siponimod involves the sphingosine-1-phosphate receptor 5. CONCLUSION: This Xenopus transgenic line constitutes a simple in vivo screening platform for myelin repair therapeutics. We validated several known promyelinating compounds and demonstrated that the strong remyelinating efficacy of siponimod implicates the sphingosine-1-phosphate receptor 5.


Assuntos
Azetidinas/farmacologia , Compostos de Benzil/farmacologia , Modelos Animais de Doenças , Receptores de Lisoesfingolipídeo/agonistas , Remielinização/efeitos dos fármacos , Animais , Animais Geneticamente Modificados , Feminino , Larva , Masculino , Remielinização/fisiologia , Xenopus
13.
Sci Rep ; 7(1): 16554, 2017 11 29.
Artigo em Inglês | MEDLINE | ID: mdl-29185448

RESUMO

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.


Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/fisiologia , Zigoto/metabolismo , Animais , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Eletroporação/métodos , Feminino , Genótipo , Microscopia Confocal , Mutação , Ratos
16.
Cell Rep ; 14(9): 2263-2272, 2016 Mar 08.
Artigo em Inglês | MEDLINE | ID: mdl-26923600

RESUMO

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.


Assuntos
Sistemas CRISPR-Cas , Endonucleases/genética , Edição de Genes , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/genética , Animais , Sequência de Bases , Linhagem Celular Tumoral , Marcação de Genes , Humanos , Mutação INDEL , Camundongos , Oligonucleotídeos/genética , Ratos , Peixe-Zebra
17.
Nucleic Acids Res ; 44(10): 4721-33, 2016 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-26921407

RESUMO

DNA ends get exposed in cells upon either normal or dysfunctional cellular processes or molecular events. Telomeres need to be protected by the shelterin complex to avoid junctions occurring between chromosomes while failing topoisomerases or clustered DNA damage processing may produce double-strand breaks, thus requiring swift repair to avoid cell death. The rigorous study of the great many proteins involved in the maintenance of DNA integrity is a challenging task because of the innumerous unspecific electrostatic and/or hydrophobic DNA-protein interactions that arise due to the chemical nature of DNA. We devised a technique that discriminates the proteins recruited specifically at DNA ends from those that bind to DNA because of a generic affinity for the double helix. Our study shows that the DNA ends proteome comprises proteins of an unexpectedly wide functional spectrum, ranging from DNA repair to ribosome biogenesis and cytoskeleton, including novel proteins of undocumented function. A global mapping of the identified proteome on published DNA repair protein networks demonstrated the excellent specificity and functional coverage of our purification technique. Finally, the native nucleoproteic complexes that assembled specifically onto DNA ends were shown to be endowed with a highly efficient DNA repair activity.


Assuntos
Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Proteoma/metabolismo , Cromatografia de Afinidade/métodos , Reparo do DNA , Células HeLa , Humanos , Nucleoproteínas/metabolismo
18.
Methods Mol Biol ; 1338: 245-59, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26443226

RESUMO

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.


Assuntos
Animais Geneticamente Modificados/genética , Endonucleases/genética , Técnicas de Inativação de Genes/métodos , Animais , Genoma , Recombinação Homóloga/genética , Humanos , RNA Mensageiro/genética , Ratos , Transativadores/genética
20.
Sci Rep ; 5: 14410, 2015 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-26442875

RESUMO

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.


Assuntos
Sistemas CRISPR-Cas/genética , Engenharia de Proteínas , Reparo de DNA por Recombinação/genética , Zigoto/fisiologia , Animais , Camundongos , Camundongos Endogâmicos C57BL , Microinjeções , Ratos , Ratos Sprague-Dawley
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