Efficient edition of the bovine PRNP prion gene in somatic cells and IVF embryos using the CRISPR/Cas9 system

Autores
Bevacqua, Romina Jimena; Fernandez-Martín, R.; Savy, Virginia; Canel, Natalia Gabriela; Gismondi, Maria Ines; Kues, Wilfried A.; Carlson, Daniel F.; Fahrenkrug, S.C.; Niemann, H.; Taboga, Oscar Alberto; Ferraris, Sergio; Salamone, Daniel Felipe
Año de publicación
2016
Idioma
inglés
Tipo de recurso
artículo
Estado
versión aceptada
Descripción
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.
Fil: Bevacqua, Romina Jimena. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Fernandez Martin, R.. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; Argentina
Fil: Savy, Virginia. Universidad de Buenos Aires. Facultad de Agronomía. Laboratorio de Biotecnología Animal; Argentina
Fil: Canel, Natalia Gabriela. Universidad de Buenos Aires. Facultad de Agronomía. Laboratorio de Biotecnología Animal; Argentina
Fil: Gismondi, Maria Ines. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina
Fil: Kues, Wilfried A. Institute of Farm Animal Genetics. Friedrich-Loeffler-Institute; Alemania
Fil: Carlson, Daniel F. Recombinetics Inc; Estados Unidos
Fil: Fahrenkrug, S.C. Recombinetics Inc; Estados Unidos
Fil: Niemann, Heiner. Institute of Farm Animal Genetics. Alemania
Fil: Taboga, Oscar Alberto. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina
Ferraris, Sergio. Universidad Maimónides. Centro de Investigación y Desarrollo en Medicina Experimental. Laboratorio de Clonación y Transgénesis; Argentina
Fil: Salamone, Daniel Felipe. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fuente
Theriogenology 86 (8) :1886-1896.e1. (November 2016)
Materia
Genética
Genetics
Somatic Cells
Cattle
Spongiform Encephalopathy
Células Somáticas
Ganado Bovino
Encefalopatía Espongiforme
Mal de la Vaca Loca
Nivel de accesibilidad
acceso restringido
Condiciones de uso
Repositorio
INTA Digital (INTA)
Institución
Instituto Nacional de Tecnología Agropecuaria
OAI Identificador
oai:localhost:20.500.12123/1111

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oai_identifier_str oai:localhost:20.500.12123/1111
network_acronym_str INTADig
repository_id_str l
network_name_str INTA Digital (INTA)
spelling Efficient edition of the bovine PRNP prion gene in somatic cells and IVF embryos using the CRISPR/Cas9 systemBevacqua, Romina JimenaFernandez-Martín, R.Savy, VirginiaCanel, Natalia GabrielaGismondi, Maria InesKues, Wilfried A.Carlson, Daniel F.Fahrenkrug, S.C.Niemann, H.Taboga, Oscar AlbertoFerraris, SergioSalamone, Daniel FelipeGenéticaGeneticsSomatic CellsCattleSpongiform EncephalopathyCélulas SomáticasGanado BovinoEncefalopatía EspongiformeMal de la Vaca LocaThe 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.Fil: Bevacqua, Romina Jimena. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Fernandez Martin, R.. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; ArgentinaFil: Savy, Virginia. Universidad de Buenos Aires. Facultad de Agronomía. Laboratorio de Biotecnología Animal; ArgentinaFil: Canel, Natalia Gabriela. Universidad de Buenos Aires. Facultad de Agronomía. Laboratorio de Biotecnología Animal; ArgentinaFil: Gismondi, Maria Ines. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Kues, Wilfried A. Institute of Farm Animal Genetics. Friedrich-Loeffler-Institute; AlemaniaFil: Carlson, Daniel F. Recombinetics Inc; Estados UnidosFil: Fahrenkrug, S.C. Recombinetics Inc; Estados UnidosFil: Niemann, Heiner. Institute of Farm Animal Genetics. AlemaniaFil: Taboga, Oscar Alberto. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFerraris, Sergio. Universidad Maimónides. Centro de Investigación y Desarrollo en Medicina Experimental. Laboratorio de Clonación y Transgénesis; ArgentinaFil: Salamone, Daniel Felipe. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina2017-09-04T12:50:38Z2017-09-04T12:50:38Z2016-11info:eu-repo/semantics/articleinfo:eu-repo/semantics/acceptedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfhttp://hdl.handle.net/20.500.12123/1111http://www.theriojournal.com/article/S0093-691X(16)30264-3/fulltext0093-691X (Print)1879-3231 (Online)https://doi.org/10.1016/j.theriogenology.2016.06.010Theriogenology 86 (8) :1886-1896.e1. (November 2016)reponame:INTA Digital (INTA)instname:Instituto Nacional de Tecnología Agropecuariaenginfo:eu-repo/semantics/restrictedAccess2025-09-29T13:44:09Zoai:localhost:20.500.12123/1111instacron:INTAInstitucionalhttp://repositorio.inta.gob.ar/Organismo científico-tecnológicoNo correspondehttp://repositorio.inta.gob.ar/oai/requesttripaldi.nicolas@inta.gob.arArgentinaNo correspondeNo correspondeNo correspondeopendoar:l2025-09-29 13:44:10.151INTA Digital (INTA) - Instituto Nacional de Tecnología Agropecuariafalse
dc.title.none.fl_str_mv Efficient edition of the bovine PRNP prion gene in somatic cells and IVF embryos using the CRISPR/Cas9 system
title Efficient edition of the bovine PRNP prion gene in somatic cells and IVF embryos using the CRISPR/Cas9 system
spellingShingle Efficient edition of the bovine PRNP prion gene in somatic cells and IVF embryos using the CRISPR/Cas9 system
Bevacqua, Romina Jimena
Genética
Genetics
Somatic Cells
Cattle
Spongiform Encephalopathy
Células Somáticas
Ganado Bovino
Encefalopatía Espongiforme
Mal de la Vaca Loca
title_short Efficient edition of the bovine PRNP prion gene in somatic cells and IVF embryos using the CRISPR/Cas9 system
title_full Efficient edition of the bovine PRNP prion gene in somatic cells and IVF embryos using the CRISPR/Cas9 system
title_fullStr Efficient edition of the bovine PRNP prion gene in somatic cells and IVF embryos using the CRISPR/Cas9 system
title_full_unstemmed Efficient edition of the bovine PRNP prion gene in somatic cells and IVF embryos using the CRISPR/Cas9 system
title_sort Efficient edition of the bovine PRNP prion gene in somatic cells and IVF embryos using the CRISPR/Cas9 system
dc.creator.none.fl_str_mv Bevacqua, Romina Jimena
Fernandez-Martín, R.
Savy, Virginia
Canel, Natalia Gabriela
Gismondi, Maria Ines
Kues, Wilfried A.
Carlson, Daniel F.
Fahrenkrug, S.C.
Niemann, H.
Taboga, Oscar Alberto
Ferraris, Sergio
Salamone, Daniel Felipe
author Bevacqua, Romina Jimena
author_facet Bevacqua, Romina Jimena
Fernandez-Martín, R.
Savy, Virginia
Canel, Natalia Gabriela
Gismondi, Maria Ines
Kues, Wilfried A.
Carlson, Daniel F.
Fahrenkrug, S.C.
Niemann, H.
Taboga, Oscar Alberto
Ferraris, Sergio
Salamone, Daniel Felipe
author_role author
author2 Fernandez-Martín, R.
Savy, Virginia
Canel, Natalia Gabriela
Gismondi, Maria Ines
Kues, Wilfried A.
Carlson, Daniel F.
Fahrenkrug, S.C.
Niemann, H.
Taboga, Oscar Alberto
Ferraris, Sergio
Salamone, Daniel Felipe
author2_role author
author
author
author
author
author
author
author
author
author
author
dc.subject.none.fl_str_mv Genética
Genetics
Somatic Cells
Cattle
Spongiform Encephalopathy
Células Somáticas
Ganado Bovino
Encefalopatía Espongiforme
Mal de la Vaca Loca
topic Genética
Genetics
Somatic Cells
Cattle
Spongiform Encephalopathy
Células Somáticas
Ganado Bovino
Encefalopatía Espongiforme
Mal de la Vaca Loca
dc.description.none.fl_txt_mv 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.
Fil: Bevacqua, Romina Jimena. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Fernandez Martin, R.. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; Argentina
Fil: Savy, Virginia. Universidad de Buenos Aires. Facultad de Agronomía. Laboratorio de Biotecnología Animal; Argentina
Fil: Canel, Natalia Gabriela. Universidad de Buenos Aires. Facultad de Agronomía. Laboratorio de Biotecnología Animal; Argentina
Fil: Gismondi, Maria Ines. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina
Fil: Kues, Wilfried A. Institute of Farm Animal Genetics. Friedrich-Loeffler-Institute; Alemania
Fil: Carlson, Daniel F. Recombinetics Inc; Estados Unidos
Fil: Fahrenkrug, S.C. Recombinetics Inc; Estados Unidos
Fil: Niemann, Heiner. Institute of Farm Animal Genetics. Alemania
Fil: Taboga, Oscar Alberto. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina
Ferraris, Sergio. Universidad Maimónides. Centro de Investigación y Desarrollo en Medicina Experimental. Laboratorio de Clonación y Transgénesis; Argentina
Fil: Salamone, Daniel Felipe. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
description 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.
publishDate 2016
dc.date.none.fl_str_mv 2016-11
2017-09-04T12:50:38Z
2017-09-04T12:50:38Z
dc.type.none.fl_str_mv info:eu-repo/semantics/article
info:eu-repo/semantics/acceptedVersion
http://purl.org/coar/resource_type/c_6501
info:ar-repo/semantics/articulo
format article
status_str acceptedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/20.500.12123/1111
http://www.theriojournal.com/article/S0093-691X(16)30264-3/fulltext
0093-691X (Print)
1879-3231 (Online)
https://doi.org/10.1016/j.theriogenology.2016.06.010
url http://hdl.handle.net/20.500.12123/1111
http://www.theriojournal.com/article/S0093-691X(16)30264-3/fulltext
https://doi.org/10.1016/j.theriogenology.2016.06.010
identifier_str_mv 0093-691X (Print)
1879-3231 (Online)
dc.language.none.fl_str_mv eng
language eng
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dc.format.none.fl_str_mv application/pdf
dc.source.none.fl_str_mv Theriogenology 86 (8) :1886-1896.e1. (November 2016)
reponame:INTA Digital (INTA)
instname:Instituto Nacional de Tecnología Agropecuaria
reponame_str INTA Digital (INTA)
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instname_str Instituto Nacional de Tecnología Agropecuaria
repository.name.fl_str_mv INTA Digital (INTA) - Instituto Nacional de Tecnología Agropecuaria
repository.mail.fl_str_mv tripaldi.nicolas@inta.gob.ar
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