Efficient One-Step Knockout by Electroporation of Ribonucleoproteins Into Zona-Intact Bovine Embryos

Frontiers in Genetics

Luiz Sergio Almeida Camargo¹  Pablo Juan Ross²  Alison L. Van Eenennaam²  Joseph R. Owen² 

[1] Brazilian Agricultural Research Corporation (Embrapa Dairy Cattle), Juiz de Fora, Brazil;Department of Animal Science, University of California, Davis, Davis, CA, United States;

关键词: embryo;    genome editing;    CRISPR;    Cas9;    OCT4 gene;   

DOI  :  10.3389/fgene.2020.570069

来源: DOAJ

【 摘 要 】

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 knock-out. 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.

【 授权许可】

Unknown