Efficient one-step knockout by electroporation of ribonucleoproteins into zona-intact bovine embryos.

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dc.contributorLUIZ SERGIO DE ALMEIDA CAMARGO, CNPGL; JOSEPH R. OWEN, University of California; ALISON L. VAN EENENNAAM, University of California; PABLO JUAN ROSS, University of California.
dc.creatorCAMARGO, L. S. de A.
dc.creatorOWEN, J. R.
dc.creatorVAN EENENNAAM, A. L.
dc.creatorROSS, P. J.
dc.date2021-08-12T13:00:45Z
dc.date2021-08-12T13:00:45Z
dc.date2021-08-12
dc.date2020
dc.date.accessioned2026-07-07T03:47:38Z
dc.descriptionSomatic 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.
dc.identifierFrontiers in Genetics, v. 11, article 570069, 2020.
dc.identifierhttp://www.alice.cnptia.embrapa.br/alice/handle/doc/1133487
dc.identifierhttps://doi.org/10.3389/fgene.2020.570069
dc.identifier.urihttp://hdl.handle.net/123456789/443025
dc.languageeng
dc.rightsopenAccess
dc.subjectCRISPR
dc.subjectGenome editing
dc.subjectBovino
dc.subjectReprodução Animal
dc.subjectEmbrião Animal
dc.subjectGenoma
dc.subjectEmbryo (animal)
dc.titleEfficient one-step knockout by electroporation of ribonucleoproteins into zona-intact bovine embryos.
dc.typeArtigo de periódico

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