Investigating agricultural and biomedical applications of genome editors in large animals
Huddart, Rachel Anne
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Large animal species, such as cattle, sheep and pigs, have great potential value to scientific research. This is due to their physiological similarity to humans, meaning they make excellent disease models in addition to their inherent agricultural value. However, the efficiency with which such animals can be created has been a critical barrier to their use in bioscience. Research into creating genetically modified large animals has not progressed as rapidly as research on smaller mammals, such as mice, for two main reasons. Firstly, technologies such as pluripotent stem cells, which are well established in rodents, are lacking for large animals. Secondly, large animals cannot produce as many offspring within a given time frame as mice or rats. This, combined with the low efficiencies and lack of precision of current transgenic methods, severely reduces the likelihood of obtaining an animal with a desired genotype within a viable amount of time. Recently, new tools known as ’genome editors’ have been developed to facilitate genetic modification of animals. The vastly enhanced efficiency of these editors in comparison to previous gene targeting methods, combined with the fact that genome editors do not require marker genes to be used, mean that creating genetically modified livestock is now far more feasible. This thesis investigates whether two types of genome editor, TALENs and CRISPR/Cas9, can be used to produce genetically modified large animals for a range of applications. Genome editors were combined with interspecific blastocyst complementation techniques to produce chimeric rodents where the haematopoietic system is partially or fully derived from the donor cells. This work was carried out with a long-term aim of producing chimeric animals which could produce human organs suitable for transplantation. Initial blastocyst complementation experiments were carried out by injecting murine ESCs into wildtype rat blastocysts. One animal resulting from these injections showed chimerism in several tissues. Further experiments were carried out using rat ESCs and mouse blastocysts which were either Runx1-/- or Rag1-/-, however no additional chimeras were identified. In addition to these experiments, TALENs and sgRNAs were designed against Runx1 and Rag1 in sheep and pigs in order to create a large animal model for future blastocyst complementation experiments. Increasing animal productivity is a key step in meeting the demands of an increasing global population and tackling future food insecurities. TALENs and sgRNAs for use in the CRISPR/ Cas9 system were created to target the myostatin gene in sheep. Myostatin is a negative regulator of muscle growth and animals which acquire natural inactivating mutations in both myostatin alleles exhibit a well-characterised double-muscled phenotype, where total muscle mass is about 20% greater than that of a wildtype animal. Embryo microinjections were carried out using both types of genome editor and two edited lambs were produced, one from each editor. The TALEN-edited lamb was mosaic for a deletion of arginine 283 which, upon further analysis of the muscle, did not appear to cause a significant phenotype. The CRISPR-edited lamb was heterozygous for a 20bp deletion, causing the formation of a premature stop codon and severe truncation of the mature myostatin protein. Based on data from other myostatin-knockout animals, including the Belgian Blue cattle breed, this truncated protein is not thought to be functional. To determine if this is indeed the case, the CRISPR-edited lamb is now part of a breeding programme to amplify the edited allele. To discover if genome editors could be applied to create disease-resistant animals, the project focused on foot and mouth disease. Through a literature search and bioinformatic analysis of the bovine and porcine proteomes, three host genes which are cleaved by the virus were identified; eIF4A1, eIF4G1 and IKBKG. TALENs were designed to bind and cut at the FMDV protease cleavage sites in all three genes in order to disrupt protease cleavage and reduce viral replication by slowing viral disruption of the host translation and innate immune response pathways. Although none of the TALENs showed any signs of activity, this thesis sets out some potential directions for future work. In conclusion, this thesis shows that, despite some technical issues, genome editors are a promising technology for the creation of genetically modified livestock.