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.

Cumulus oocyte complexes were aspirated from 3-5 mm follicles on pig ovaries obtained from a commercial abbatoir, rinsed and matured in vitro for 40-44 hours at 38.5 ^o C. The 100 μL drop containing oocytes and approximately 1000 washed, partially capacitated sperm/oocyte was incubated for 5-6 hours in 5% CO₂ in air. Zygotes were electroporated in Opti-MEM™ (Gibco) with the CRISPR guide and Cas9 protein 9 hours after insemination. Following culture of electroporated zygotes to the blastocyst stage, individual blastocysts were lysed in 10 µL of QE09050 buffer (Biosearch Technologies, UK), vortexed, centrifuged, and incubated at 65°C (6 minutes) and 98°C (2 minutes) followed by storage at -17^o C. DNA was amplified during nested rounds (2) of PCR, the PCR products were separated on a 0.8% agarose gel, bands excised, DNA was extracted (QIAquick Gel Extraction Kit, Qiagen #28704) and submitted for sequencing. Sequences were aligned with SnapGeneâ using the Sus Scrofa 11.1 genome. The sequences around the target sites of the two CRISPR guides from 9 representative blastocysts are presented in Figure 1. With electroporation with the combination of the two CRISPR guides targeting exon 2 of the porcine AR (targets outlined in red in the reference sequence), the vast majority of blastocysts have large deletions and a blastocyst has a 6 bp deletion. The last edit might only eliminate two amino acids in the transcript or potentially alter a third amino acid as well. The diversity of edits in the blastocysts with the majority being large deletions accurately forecasts the diversity of edits in fetuses, again with frequent large deletions observed.  

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