Efficient edition of the bovine PRNP prion gene in somatic cells and IVF embryos using the CRISPR/Cas9 system
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 manne...
| Main Authors: | , , , , , , , , , , , |
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| Format: | Artículo |
| Language: | Inglés |
| Published: |
2017
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| Subjects: | |
| Online Access: | 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 |
| _version_ | 1855482830313750528 |
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| author | 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_browse | Bevacqua, Romina Jimena Canel, Natalia Gabriela Carlson, Daniel F. Fahrenkrug, S.C. Fernandez-Martín, R. Ferraris, Sergio Gismondi, Maria Ines Kues, Wilfried A. Niemann, H. Salamone, Daniel Felipe Savy, Virginia Taboga, Oscar Alberto |
| 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_sort | Bevacqua, Romina Jimena |
| collection | INTA Digital |
| 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. |
| format | Artículo |
| id | INTA1111 |
| institution | Instituto Nacional de Tecnología Agropecuaria (INTA -Argentina) |
| language | Inglés |
| publishDate | 2017 |
| publishDateRange | 2017 |
| publishDateSort | 2017 |
| record_format | dspace |
| spelling | INTA11112018-08-24T18:01:35Z Efficient edition of the bovine PRNP prion gene in somatic cells and IVF embryos using the CRISPR/Cas9 system 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 Genética Genetics Somatic Cells Cattle Spongiform Encephalopathy Células Somáticas Ganado Bovino Encefalopatía Espongiforme Mal de la Vaca Loca 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 2017-09-04T12:50:38Z 2017-09-04T12:50:38Z 2016-11 info:ar-repo/semantics/artículo info:eu-repo/semantics/article info:eu-repo/semantics/acceptedVersion 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 eng info:eu-repo/semantics/restrictedAccess application/pdf Theriogenology 86 (8) :1886-1896.e1. (November 2016) |
| spellingShingle | Genética Genetics Somatic Cells Cattle Spongiform Encephalopathy Células Somáticas Ganado Bovino Encefalopatía Espongiforme Mal de la Vaca Loca 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 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 |
| 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_short | 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 |
| topic | Genética Genetics Somatic Cells Cattle Spongiform Encephalopathy Células Somáticas Ganado Bovino Encefalopatía Espongiforme Mal de la Vaca Loca |
| 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 |
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