Using Lentivirus and CRISPR to Modify Cattle Embryonic Genes

Developing transgenic livestock has become popular in recent years after advances in the field of genetic editing. Cattle are one of the main exports in New Zealand and are a prime target for new genetic editing tools. Applications of genetic editing in cattle can extend to increases in production, and elimination of disease genes. Due to its ease of use, CRISPR/Cas9 has become one of the most popular methods of editing genes, hence this was employed in the research. Cattle embryos in culture are very sensitive to environmental changes and for this reason, a delivery vector is necessary to deliver the genetic material as traditional transfection methods cause high rates of embryo death. The zona pellucida, a glycoprotein coat surrounding the embryo, acts as a protective agent against viral vectors, and needed to be considered in the research. This research aimed to create a novel, high titer lentivirus particle capable of transducing bovine embryos, and causing subsequent genetic modification by integration of CRISPR/Cas9 into the genome. Using fluorescent reporters, viral transduction was monitored. The research found that after optimizing transfection protocols, high-titer lentiviral particles can be produced and can infect bovine embryos. Zona pellucida removal experiments revealed over-digestion in early stage embryos, however, this was not observed in compact morulas. Removing the zona allowed for successful transduction of bovine embryos, resulting in transgenic cells expressing eGFP. While CRISPR/Cas9 experiments were in preliminary stages, these indicated eGFP knock-out in certain eGFP-HEK293T cells. Though challenges were encountered throughout the research process, solutions were explored, and it was shown that transgenic bovine embryos using lentiviral gene delivery can be produced. This indicates the high likelihood that CRISPR/Cas9 systems can be delivered this way, inducing targeted genetic modification.