Edits of AR in porcine blastocysts after CRISPR Cas9 editing of zygotes

The CRISPR Cas9 technology offers an opportunity to evaluate physiological responses to deletion of individual genes in livestock species, expanding our understanding of animal biology. Testing efficacy of individual guides used for gene deletion is desirable before the time-consuming embryo transfers planned to generate the desired model animals. These experiments were designed to test efficacy of guides to delete expression of the androgen receptor (AR) in pig zygotes and to characterize the resulting deletions. Two guides targeting exon 2 (Ensembl Sus Scrofa 11.1) were designed. Following electroporation of these guides into in vitro generated porcine zygotes, embryos were cultured to the blastocyst stage and the DNA sequenced. Over 80% of blastocysts produced following electroporation of the two guides targeting exon 2 had deletions. Deletions were biallelic. Although blastocysts might contain edits from only one of the guides, the majority of blastocysts had large deletions resulting from the combined effect of both guides.