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Registro Completo |
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Biblioteca(s): |
Embrapa Gado de Leite. |
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Data corrente: |
12/08/2021 |
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Data da última atualização: |
12/08/2021 |
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Tipo da produção científica: |
Artigo em Periódico Indexado |
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Autoria: |
CAMARGO, L. S. de A.; OWEN, J. R.; VAN EENENNAAM, A. L.; ROSS, P. J. |
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Afiliação: |
LUIZ SERGIO DE ALMEIDA CAMARGO, CNPGL; JOSEPH R. OWEN, University of California; ALISON L. VAN EENENNAAM, University of California; PABLO JUAN ROSS, University of California. |
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Título: |
Efficient one-step knockout by electroporation of ribonucleoproteins into zona-intact bovine embryos. |
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Ano de publicação: |
2020 |
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Fonte/Imprenta: |
Frontiers in Genetics, v. 11, article 570069, 2020. |
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DOI: |
https://doi.org/10.3389/fgene.2020.570069 |
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Idioma: |
Inglês |
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Conteúdo: |
Somatic cell nuclear transfer or cytoplasm microinjection have been used to generate genome-edited farm animals; however, these methods have several drawbacks that reduce their efficiency. This study aimed to develop electroporation conditions that allow delivery of CRISPR/Cas9 system to bovine zygotes for efficient gene knockout. We optimized electroporation conditions to deliver Cas9:sgRNA ribonucleoproteins to bovine zygotes without compromising embryo development. Higher electroporation pulse voltage resulted in increased membrane permeability; however, voltages above 15 V/mm decreased embryo developmental potential. The zona pellucida of bovine embryos was not a barrier to efficient RNP electroporation. Using parameters optimized for maximal membrane permeability while maintaining developmental competence we achieved high rates of gene editing when targeting bovine OCT4, which resulted in absence of OCT4 protein in 100% of the evaluated embryos and the expected arrest of embryonic development at the morula stage. In conclusion, Cas9:sgRNA ribonucleoproteins can be delivered efficiently by electroporation to zona-intact bovine zygotes, resulting in efficient gene knockouts. |
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Palavras-Chave: |
CRISPR; Genome editing. |
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Thesagro: |
Bovino; Embrião Animal; Genoma; Reprodução Animal. |
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Thesaurus Nal: |
Embryo (animal). |
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Categoria do assunto: |
L Ciência Animal e Produtos de Origem Animal |
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URL: |
https://ainfo.cnptia.embrapa.br/digital/bitstream/item/225093/1/Efficient-one-step.pdf
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Marc: |
LEADER 01958naa a2200253 a 4500
001 2133487
005 2021-08-12
008 2020 bl uuuu u00u1 u #d
024 7 $ahttps://doi.org/10.3389/fgene.2020.570069$2DOI
100 1 $aCAMARGO, L. S. de A.
245 $aEfficient one-step knockout by electroporation of ribonucleoproteins into zona-intact bovine embryos.$h[electronic resource]
260 $c2020
520 $aSomatic cell nuclear transfer or cytoplasm microinjection have been used to generate genome-edited farm animals; however, these methods have several drawbacks that reduce their efficiency. This study aimed to develop electroporation conditions that allow delivery of CRISPR/Cas9 system to bovine zygotes for efficient gene knockout. We optimized electroporation conditions to deliver Cas9:sgRNA ribonucleoproteins to bovine zygotes without compromising embryo development. Higher electroporation pulse voltage resulted in increased membrane permeability; however, voltages above 15 V/mm decreased embryo developmental potential. The zona pellucida of bovine embryos was not a barrier to efficient RNP electroporation. Using parameters optimized for maximal membrane permeability while maintaining developmental competence we achieved high rates of gene editing when targeting bovine OCT4, which resulted in absence of OCT4 protein in 100% of the evaluated embryos and the expected arrest of embryonic development at the morula stage. In conclusion, Cas9:sgRNA ribonucleoproteins can be delivered efficiently by electroporation to zona-intact bovine zygotes, resulting in efficient gene knockouts.
650 $aEmbryo (animal)
650 $aBovino
650 $aEmbrião Animal
650 $aGenoma
650 $aReprodução Animal
653 $aCRISPR
653 $aGenome editing
700 1 $aOWEN, J. R.
700 1 $aVAN EENENNAAM, A. L.
700 1 $aROSS, P. J.
773 $tFrontiers in Genetics$gv. 11, article 570069, 2020.
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Embrapa Gado de Leite (CNPGL) |
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