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

• Autor(a) principal: CAMARGO, L. S. de A.
• Data de Publicação: 2020
• Outros Autores: OWEN, J. R., VAN EENENNAAM, A. L., ROSS, P. J.
• Tipo de documento: Artigo
• Idioma: eng
• Título da fonte: Repositório Institucional da EMBRAPA (Repository Open Access to Scientific Information from EMBRAPA - Alice)
• Texto Completo: http://www.alice.cnptia.embrapa.br/alice/handle/doc/1133487; https://doi.org/10.3389/fgene.2020.570069
• topic: CRISPR; Genome editing; Bovino; Reprodução Animal; Embrião Animal; Genoma; Embryo (animal)

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.