Generation of myostatin edited horse embryos using CRISPR/Cas9 technology and somatic cell nuclear transfer

Autores
Moro, Lucía Natalia; Viale, Diego Luis; Bastón, Juan Ignacio; Arnold, Victoria; Suvá, Mariana; Wiedenmann, Elisabet; Olguín, Martín; Miriuka, Santiago Gabriel; Vichera, Gabriel Damian
Año de publicación
2020
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
The application of new technologies for gene editing in horses may allow the generation of improved sportive individuals.Here, we aimed to knock out the myostatin gene (MSTN), a negative regulator of muscle mass development, using CRISPR/Cas9 and to generate edited embryos for the first time in horses. We nucleofected horse fetal fibroblasts with 1, 2 or 5 µg of 2 different gRNA/Cas9 plasmids targeting the first exon of MSTN. We observed that increasing plasmid concentrations improved mutation efficiency. The average efficiency was 63.6% for gRNA1 (14/22 edited clonal cell lines) and 96.2% for gRNA2 (25/26 edited clonal cell lines). Three clonal cell lines were chosen for embryo generation by somatic cell nuclear transfer: one with a monoallelic edition, one with biallelic heterozygous editions and one with a biallelic homozygous edition, which rendered edited blastocysts in each case. Both MSTN editions and off-targets were analyzed in the embryos. In conclusion, CRISPR/Cas9 proved an efficient method to edit the horse genome in a dose dependent manner with high specificity. Adapting this technology sport advantageous alleles could be generated, and a precision breeding program could be developed.
Fil: Moro, Lucía Natalia. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Viale, Diego Luis. Universidad Nacional de San Martín. Escuela de Ciencia y Tecnología. Centro de Estudios en Salud y Medio Ambiente; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Bastón, Juan Ignacio. Kheiron Sa.; Argentina
Fil: Arnold, Victoria. Kheiron Sa.; Argentina
Fil: Suvá, Mariana. Kheiron Sa.; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Wiedenmann, Elisabet. Kheiron Sa.; Argentina
Fil: Olguín, Martín. Kheiron Sa.; Argentina
Fil: Miriuka, Santiago Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; Argentina
Fil: Vichera, Gabriel Damian. Kheiron Sa.; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Materia
CRISPR-Cas9
Gene Edition
SCNT
Embryos
Nivel de accesibilidad
acceso abierto
Condiciones de uso
https://creativecommons.org/licenses/by/2.5/ar/
Repositorio
CONICET Digital (CONICET)
Institución
Consejo Nacional de Investigaciones Científicas y Técnicas
OAI Identificador
oai:ri.conicet.gov.ar:11336/171450
Acceder
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oai_identifier_str oai:ri.conicet.gov.ar:11336/171450
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling Generation of myostatin edited horse embryos using CRISPR/Cas9 technology and somatic cell nuclear transferMoro, Lucía NataliaViale, Diego LuisBastón, Juan IgnacioArnold, VictoriaSuvá, MarianaWiedenmann, ElisabetOlguín, MartínMiriuka, Santiago GabrielVichera, Gabriel DamianCRISPR-Cas9Gene EditionSCNTEmbryoshttps://purl.org/becyt/ford/1.6https://purl.org/becyt/ford/1https://purl.org/becyt/ford/4.4https://purl.org/becyt/ford/4https://purl.org/becyt/ford/1.6https://purl.org/becyt/ford/1The application of new technologies for gene editing in horses may allow the generation of improved sportive individuals.Here, we aimed to knock out the myostatin gene (MSTN), a negative regulator of muscle mass development, using CRISPR/Cas9 and to generate edited embryos for the first time in horses. We nucleofected horse fetal fibroblasts with 1, 2 or 5 µg of 2 different gRNA/Cas9 plasmids targeting the first exon of MSTN. We observed that increasing plasmid concentrations improved mutation efficiency. The average efficiency was 63.6% for gRNA1 (14/22 edited clonal cell lines) and 96.2% for gRNA2 (25/26 edited clonal cell lines). Three clonal cell lines were chosen for embryo generation by somatic cell nuclear transfer: one with a monoallelic edition, one with biallelic heterozygous editions and one with a biallelic homozygous edition, which rendered edited blastocysts in each case. Both MSTN editions and off-targets were analyzed in the embryos. In conclusion, CRISPR/Cas9 proved an efficient method to edit the horse genome in a dose dependent manner with high specificity. Adapting this technology sport advantageous alleles could be generated, and a precision breeding program could be developed.Fil: Moro, Lucía Natalia. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Viale, Diego Luis. Universidad Nacional de San Martín. Escuela de Ciencia y Tecnología. Centro de Estudios en Salud y Medio Ambiente; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Bastón, Juan Ignacio. Kheiron Sa.; ArgentinaFil: Arnold, Victoria. Kheiron Sa.; ArgentinaFil: Suvá, Mariana. Kheiron Sa.; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Wiedenmann, Elisabet. Kheiron Sa.; ArgentinaFil: Olguín, Martín. Kheiron Sa.; ArgentinaFil: Miriuka, Santiago Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; ArgentinaFil: Vichera, Gabriel Damian. Kheiron Sa.; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaNature2020-09info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/171450Moro, Lucía Natalia; Viale, Diego Luis; Bastón, Juan Ignacio; Arnold, Victoria; Suvá, Mariana; et al.; Generation of myostatin edited horse embryos using CRISPR/Cas9 technology and somatic cell nuclear transfer; Nature; Scientific Reports; 10; 1; 9-2020; 1-152045-2322CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/url/http://www.nature.com/articles/s41598-020-72040-4info:eu-repo/semantics/altIdentifier/doi/10.1038/s41598-020-72040-4info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-09-29T10:33:23Zoai:ri.conicet.gov.ar:11336/171450instacron:CONICETInstitucionalhttp://ri.conicet.gov.ar/Organismo científico-tecnológicoNo correspondehttp://ri.conicet.gov.ar/oai/requestdasensio@conicet.gov.ar; lcarlino@conicet.gov.arArgentinaNo correspondeNo correspondeNo correspondeopendoar:34982025-09-29 10:33:24.037CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv Generation of myostatin edited horse embryos using CRISPR/Cas9 technology and somatic cell nuclear transfer
title Generation of myostatin edited horse embryos using CRISPR/Cas9 technology and somatic cell nuclear transfer
spellingShingle Generation of myostatin edited horse embryos using CRISPR/Cas9 technology and somatic cell nuclear transfer
Moro, Lucía Natalia
CRISPR-Cas9
Gene Edition
SCNT
Embryos
title_short Generation of myostatin edited horse embryos using CRISPR/Cas9 technology and somatic cell nuclear transfer
title_full Generation of myostatin edited horse embryos using CRISPR/Cas9 technology and somatic cell nuclear transfer
title_fullStr Generation of myostatin edited horse embryos using CRISPR/Cas9 technology and somatic cell nuclear transfer
title_full_unstemmed Generation of myostatin edited horse embryos using CRISPR/Cas9 technology and somatic cell nuclear transfer
title_sort Generation of myostatin edited horse embryos using CRISPR/Cas9 technology and somatic cell nuclear transfer
dc.creator.none.fl_str_mv Moro, Lucía Natalia
Viale, Diego Luis
Bastón, Juan Ignacio
Arnold, Victoria
Suvá, Mariana
Wiedenmann, Elisabet
Olguín, Martín
Miriuka, Santiago Gabriel
Vichera, Gabriel Damian
author Moro, Lucía Natalia
author_facet Moro, Lucía Natalia
Viale, Diego Luis
Bastón, Juan Ignacio
Arnold, Victoria
Suvá, Mariana
Wiedenmann, Elisabet
Olguín, Martín
Miriuka, Santiago Gabriel
Vichera, Gabriel Damian
author_role author
author2 Viale, Diego Luis
Bastón, Juan Ignacio
Arnold, Victoria
Suvá, Mariana
Wiedenmann, Elisabet
Olguín, Martín
Miriuka, Santiago Gabriel
Vichera, Gabriel Damian
author2_role author
author
author
author
author
author
author
author
dc.subject.none.fl_str_mv CRISPR-Cas9
Gene Edition
SCNT
Embryos
topic CRISPR-Cas9
Gene Edition
SCNT
Embryos
purl_subject.fl_str_mv https://purl.org/becyt/ford/1.6
https://purl.org/becyt/ford/1
https://purl.org/becyt/ford/4.4
https://purl.org/becyt/ford/4
https://purl.org/becyt/ford/1.6
https://purl.org/becyt/ford/1
dc.description.none.fl_txt_mv The application of new technologies for gene editing in horses may allow the generation of improved sportive individuals.Here, we aimed to knock out the myostatin gene (MSTN), a negative regulator of muscle mass development, using CRISPR/Cas9 and to generate edited embryos for the first time in horses. We nucleofected horse fetal fibroblasts with 1, 2 or 5 µg of 2 different gRNA/Cas9 plasmids targeting the first exon of MSTN. We observed that increasing plasmid concentrations improved mutation efficiency. The average efficiency was 63.6% for gRNA1 (14/22 edited clonal cell lines) and 96.2% for gRNA2 (25/26 edited clonal cell lines). Three clonal cell lines were chosen for embryo generation by somatic cell nuclear transfer: one with a monoallelic edition, one with biallelic heterozygous editions and one with a biallelic homozygous edition, which rendered edited blastocysts in each case. Both MSTN editions and off-targets were analyzed in the embryos. In conclusion, CRISPR/Cas9 proved an efficient method to edit the horse genome in a dose dependent manner with high specificity. Adapting this technology sport advantageous alleles could be generated, and a precision breeding program could be developed.
Fil: Moro, Lucía Natalia. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Viale, Diego Luis. Universidad Nacional de San Martín. Escuela de Ciencia y Tecnología. Centro de Estudios en Salud y Medio Ambiente; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Bastón, Juan Ignacio. Kheiron Sa.; Argentina
Fil: Arnold, Victoria. Kheiron Sa.; Argentina
Fil: Suvá, Mariana. Kheiron Sa.; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Wiedenmann, Elisabet. Kheiron Sa.; Argentina
Fil: Olguín, Martín. Kheiron Sa.; Argentina
Fil: Miriuka, Santiago Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; Argentina
Fil: Vichera, Gabriel Damian. Kheiron Sa.; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
description The application of new technologies for gene editing in horses may allow the generation of improved sportive individuals.Here, we aimed to knock out the myostatin gene (MSTN), a negative regulator of muscle mass development, using CRISPR/Cas9 and to generate edited embryos for the first time in horses. We nucleofected horse fetal fibroblasts with 1, 2 or 5 µg of 2 different gRNA/Cas9 plasmids targeting the first exon of MSTN. We observed that increasing plasmid concentrations improved mutation efficiency. The average efficiency was 63.6% for gRNA1 (14/22 edited clonal cell lines) and 96.2% for gRNA2 (25/26 edited clonal cell lines). Three clonal cell lines were chosen for embryo generation by somatic cell nuclear transfer: one with a monoallelic edition, one with biallelic heterozygous editions and one with a biallelic homozygous edition, which rendered edited blastocysts in each case. Both MSTN editions and off-targets were analyzed in the embryos. In conclusion, CRISPR/Cas9 proved an efficient method to edit the horse genome in a dose dependent manner with high specificity. Adapting this technology sport advantageous alleles could be generated, and a precision breeding program could be developed.
publishDate 2020
dc.date.none.fl_str_mv 2020-09
dc.type.none.fl_str_mv info:eu-repo/semantics/article
info:eu-repo/semantics/publishedVersion
http://purl.org/coar/resource_type/c_6501
info:ar-repo/semantics/articulo
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/11336/171450
Moro, Lucía Natalia; Viale, Diego Luis; Bastón, Juan Ignacio; Arnold, Victoria; Suvá, Mariana; et al.; Generation of myostatin edited horse embryos using CRISPR/Cas9 technology and somatic cell nuclear transfer; Nature; Scientific Reports; 10; 1; 9-2020; 1-15
2045-2322
CONICET Digital
CONICET
url http://hdl.handle.net/11336/171450
identifier_str_mv Moro, Lucía Natalia; Viale, Diego Luis; Bastón, Juan Ignacio; Arnold, Victoria; Suvá, Mariana; et al.; Generation of myostatin edited horse embryos using CRISPR/Cas9 technology and somatic cell nuclear transfer; Nature; Scientific Reports; 10; 1; 9-2020; 1-15
2045-2322
CONICET Digital
CONICET
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/url/http://www.nature.com/articles/s41598-020-72040-4
info:eu-repo/semantics/altIdentifier/doi/10.1038/s41598-020-72040-4
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
https://creativecommons.org/licenses/by/2.5/ar/
eu_rights_str_mv openAccess
rights_invalid_str_mv https://creativecommons.org/licenses/by/2.5/ar/
dc.format.none.fl_str_mv application/pdf
application/pdf
dc.publisher.none.fl_str_mv Nature
publisher.none.fl_str_mv Nature
dc.source.none.fl_str_mv reponame:CONICET Digital (CONICET)
instname:Consejo Nacional de Investigaciones Científicas y Técnicas
reponame_str CONICET Digital (CONICET)
collection CONICET Digital (CONICET)
instname_str Consejo Nacional de Investigaciones Científicas y Técnicas
repository.name.fl_str_mv CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicas
repository.mail.fl_str_mv dasensio@conicet.gov.ar; lcarlino@conicet.gov.ar
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score 13.070432

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