Gene Knockouts in Goats Using CRISPR/Cas9 System and Somatic Cell Nuclear Transfer.

The combination of CRISPR/Cas9 and SCNT techniques greatly facilitates the production of gene-edited livestock. Here, we describe the detailed procedure to create gene knockout goats using this strategy starting from the construction of CRISPR/Cas9 targeting vectors to the transfer of cloned embryos into recipient females. In this procedure, the transfection conditions for goat fibroblasts were optimized due to their high sensitivity to electrotransfection, which enabled the isolation of single-cell colonies carrying simultaneous disruption of multiple genes for SCNT with a single co-transfection of pooled CRISPR/Cas9 targeting vectors.