Date of Award:

5-2020

Document Type:

Thesis

Degree Name:

Master of Science (MS)

Department:

Animal, Dairy, and Veterinary Sciences

Committee Chair(s)

Zhongde Wang (Committee Co-Chair), Dirk K. Vanderwall (Committee Co-Chair)

Committee

Zhongde Wang

Committee

Dirk K. Vanderwall

Committee

Mirella Meyer-Ficca

Abstract

HERDA (Heritable Equine Regional Dermal Asthenia) is a genetic skin disease mainly found in Quarter Horses, but also in Appaloosa and American Paint breeds. HERDA is similar to Ehlers-Danlos syndrome in humans, with symptoms including stretchy skin, hyperflexible joints, and, unique to HERDA equine, spontaneous skin sloughing. Horses affected by HERDA are not suitable for performing and are oftentimes euthanized. Some carriers for the HERDA-mutation are very competitive in the American Quarter Horse industry, especially in cutting events where it is believed, yet unproven, to give them an advantage with increased flexibility. It is also possible that the genomic locus (or loci) that links to the competitive performance traits is located close to the HERDA-causing mutation, which could lead to the co-segregation of this performance trait with the HERDA-causing mutation.

Direct-line breeding strategies in the last 30 years have increased the number of HERDA-affected equine causing this disease to increase in frequency among the Quarter Horse breed. Since no treatment exists for HERDA, owners often heavily invest in HERDA horses before the symptoms arise at around two years of age. These horses are often euthanized to alleviate pain and stress on the horse and to mitigate the costly upkeep by the owner.

HERDA-affected horses carry a homozygous single nucleotide mutation (c.115 G > A) in exon 1 of peptidyl-prolyl Isomerase B (PPIB). Gene editing approaches would be preferable for correcting this genetic disease, since it can precisely correct the mutation without altering any other genetic traits in the elite horse breeds that have been heavily selected for. By employing the CRISPR/Cas9 system, we have sought to correct the HERDA-causing mutation in the PPIB gene. The CRISPR/Cas9 system is comprised of a bacterial endonuclease protein called Cas9 and a guide RNA sequence to direct Cas9 to target the genome in a sequence-specific manner by introducing DNA double-strand breaks (DSBs). The introduction of DNA DSBs promotes the activation and recruitment of homologous recombination (HR)-mediated DNA repair machineries to repair the broken DNA; if oligonucleotides with the desired DNA sequence are co-delivered with the CRISPR/Cas9 system into cells, the HR-mediated DNA repair mechanism can replace the targeted sequence in the genome with the oligonucleotide’s sequence, therefore, achieving gene correction or editing. We designed sgRNAs to target genomic sequences in close vicinity of the HERDA-causing mutation and a single-stranded DNA oligonucleotide containing the normal (wild type) PPIB genotype. Co-delivery of the CRISPR/Cas9/sgRNA complex with the donor oligonucleotide has successfully led to the production of gene edited cells. We established single-cell derived colonies from the edited cells and achieved 7.3% monoallelicand 2.4% biallelic editing frequencies. The gene edited fibroblasts were cryopreserved as an initial step for future HERDA-free equine cloning projects to develop the first gene edited horses.

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