Therapeutic Genome Editing of Complex Vertebral Malformation in Cattle

Abstract

Autosomal recessive genetic disorders such as Complex Vertebral Malformation (CVM) cause a significant economic burden to dairy producers and impede genetic progress in the dairy industry as a whole. Many of these diseases, including CVM, have homozygous lethal phenotypes, and thus negatively impact the fertility and breeding value of heterozygous carriers. Identification of carriers typically results in culling, and forfeiture of the animal’s genetic value irrespective of the lethal recessive. Genome engineering technologies provide an opportunity to rescue the genetic value of carrier animals with economically significant production traits by repairing the disease-causing alleles. This thesis describes the optimization of a workflow for the correction of bovine CVM via SNP modification in primary cells using the CRISPR Cas9 system and ssDNA donor templates. It also attempts to quantify differences in the efficiency of SNP modifications between delivery methods for CRISPR, as well as the location of CRISPR cutting with regard to the mutation, and the length of ssDNA donor homology arms.