An electrochemical protocol for CRISPR-mediated gene-editing of sheep embryonic fibroblast cells

Genetic engineering of farm animals is commonly carried out via cell-mediated transfection followed by somatic cell nuclear transfer. However, efficient transfer of exogenous DNA into ovine embryonic fibroblast (EF) cells without compromising cell viability have remained a challenging issue. Here, we aimed to develop a protocol for electrotransfection of sheep EF cells. First, we optimized the pulsing condition using an OptiMEM-GlutaMAX medium as the electroporation buffer and found two pulses of 270 V, each for 10 ms and 10 s interval, is the most efficient condition to have a high rate of transfection and cell survival. Moreover, supplementing 3 % dimethyl sulfoxide (DMSO) into the electroporation medium considerably improved the cell viability after the electroporation process. The electroporation procedure resulted in > 98% transfection efficiency and > 97 % cell survival rate using reporter plasmids. Finally, using CRISPR/Cas9-encoding vectors, we targeted BMP15 and GDF9 genes in sheep EF cells. The electroporated cells are associated with a 52 % indels rate using single gRNAs as well as a highly efficient target deletion using two gRNAs. In conclusion, we developed an electrotransfection protocol using the OptiMEM-GlutaMAX medium supplemented with 3 % DMSO for sheep EF cells. The electroporation method can be used for cell-mediated gene-editing in sheep.


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