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dc.contributor.author
Bevacqua, Romina Jimena  
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Fernández y Martín, Rafael  
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Savy, Virginia  
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Canel, Natalia Gabriela  
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Gismondi, Maria Ines  
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Kues, W. A.  
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Carlson, D. F.  
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Fahrenkrug, S. C.  
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Niemann, H.  
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Taboga, Oscar Alberto  
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Ferraris, S.  
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Salamone, Daniel Felipe  
dc.date.available
2018-07-06T15:52:50Z  
dc.date.issued
2016-11  
dc.identifier.citation
Bevacqua, Romina Jimena; Fernández y Martín, Rafael; Savy, Virginia; Canel, Natalia Gabriela; Gismondi, Maria Ines; et al.; Efficient edition of the bovine PRNP prion gene in somatic cells and IVF embryos using the CRISPR/Cas9 system; Elsevier Science Inc; Theriogenology; 86; 8; 11-2016; 1886-1896  
dc.identifier.issn
0093-691X  
dc.identifier.uri
http://hdl.handle.net/11336/51499  
dc.description.abstract
The recently developed engineered nucleases, such as zinc-finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated nuclease (Cas) 9, provide new opportunities for gene editing in a straightforward manner. However, few reports are available regarding CRISPR application and efficiency in cattle. Here, the CRISPR/Cas9 system was used with the aim of inducing knockout and knock-in alleles of the bovine PRNP gene, responsible for mad cow disease, both in bovine fetal fibroblasts and in IVF embryos. Five single-guide RNAs were designed to target 875 bp of PRNP exon 3, and all five were codelivered with Cas9. The feasibility of inducing homologous recombination (HR) was evaluated with a reporter vector carrying EGFP flanked by 1 kbp PRNP regions (pHRegfp). For somatic cells, plasmids coding for Cas9 and for each of the five single-guide RNAs (pCMVCas9 and pSPgRNAs) were transfected under two different conditions (1X and 2X). For IVF zygotes, cytoplasmic injection was conducted with either plasmids or mRNA. For plasmid injection groups, 1 pg pCMVCas9 + 0.1 pg of each pSPgRNA (DNA2X) was used per zygote. In the case of RNA, two amounts (RNA1X and RNA2X) were compared. To assess the occurrence of HR, a group additionally cotransfected or coinjected with pHRegfp plasmid was included. Somatic cell lysates were analyzed by polymerase chain reaction and surveyor assay. In the case of embryos, the in vitro development and the genotype of blastocysts were evaluated by polymerase chain reaction and sequencing. In somatic cells, 2X transfection resulted in indels and large deletions of the targeted PRNP region. Regarding embryo injection, higher blastocyst rates were obtained for RNA injected groups (46/103 [44.6%] and 55/116 [47.4%] for RNA1X and RNA2X) than for the DNA2X group (26/140 [18.6%], P < 0.05). In 46% (26/56) of the total sequenced blastocysts, specific gene editing was detected. The total number of genetic modifications (29) was higher than the total number of gene-edited embryos, as three blastocysts from the group RNA2X reported more than one type of modification. The modifications included indels (10/56; 17.9%) and large deletions (19/56; 33.9%). Moreover, it was possible to detect HR in 1/8 (12.5%) embryos treated with RNA2X. These results report that the CRISPR/Cas9 system can be applied for site-specific edition of the bovine genome, which could have a great impact on the development of large animals resistant to important zoonotic diseases.  
dc.format
application/pdf  
dc.language.iso
eng  
dc.publisher
Elsevier Science Inc  
dc.rights
info:eu-repo/semantics/openAccess  
dc.rights.uri
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/  
dc.subject
Bovine Spongiform Encephalopathy  
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Cattle  
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Engineered Nuclease  
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Gene Targeting  
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Mad Cow Disease  
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Ruminant  
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Otras Ciencias Biológicas  
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Ciencias Biológicas  
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CIENCIAS NATURALES Y EXACTAS  
dc.title
Efficient edition of the bovine PRNP prion gene in somatic cells and IVF embryos using the CRISPR/Cas9 system  
dc.type
info:eu-repo/semantics/article  
dc.type
info:ar-repo/semantics/artículo  
dc.type
info:eu-repo/semantics/publishedVersion  
dc.date.updated
2018-06-22T14:37:05Z  
dc.journal.volume
86  
dc.journal.number
8  
dc.journal.pagination
1886-1896  
dc.journal.pais
Estados Unidos  
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Los Altos  
dc.description.fil
Fil: Bevacqua, Romina Jimena. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Unidad Ejecutora de Investigaciones en Producción Animal. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Unidad Ejecutora de Investigaciones en Producción Animal; Argentina  
dc.description.fil
Fil: Fernández y Martín, Rafael. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Unidad Ejecutora de Investigaciones en Producción Animal. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Unidad Ejecutora de Investigaciones en Producción Animal; Argentina  
dc.description.fil
Fil: Savy, Virginia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Unidad Ejecutora de Investigaciones en Producción Animal. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Unidad Ejecutora de Investigaciones en Producción Animal; Argentina  
dc.description.fil
Fil: Canel, Natalia Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Unidad Ejecutora de Investigaciones en Producción Animal. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Unidad Ejecutora de Investigaciones en Producción Animal; Argentina  
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Fil: Gismondi, Maria Ines. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina  
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Fil: Kues, W. A.. Institute of Farm Animal Genetics; Alemania  
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Fil: Carlson, D. F.. Recombinetics Inc.; Estados Unidos  
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Fil: Fahrenkrug, S. C.. Recombinetics Inc.; Estados Unidos  
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Fil: Niemann, H.. Institute of Farm Animal Genetics; Alemania  
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Fil: Taboga, Oscar Alberto. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina  
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Fil: Ferraris, S.. Universidad Maimónides; Argentina  
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Fil: Salamone, Daniel Felipe. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Unidad Ejecutora de Investigaciones en Producción Animal. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Unidad Ejecutora de Investigaciones en Producción Animal; Argentina  
dc.journal.title
Theriogenology  
dc.relation.alternativeid
info:eu-repo/semantics/altIdentifier/doi/https://dx.doi.org/10.1016/j.theriogenology.2016.06.010  
dc.relation.alternativeid
info:eu-repo/semantics/altIdentifier/url/https://www.sciencedirect.com/science/article/pii/S0093691X16302643