Genome engineering in large animals for agricultural and biomedical applications

Precision genetics will enhance genome-based improvement of livestock for agriculture and biomedicine. This thesis aimed to modify large animal genomes with precision; as the technologies progressed, our capability expanded from random insertional transgenesis to nucleotide-level precision.;It began with Sleeping Beauty (SB) transposon mediated rapid integration of dominant negative Myostatin alleles. All piglets generated from treated cells harbored the transgenes; however, we were unable to study phenotypes due to death of the founder animals. We then sought to introgress a SNP into porcine Myostatin through recombinant Adeno-associated Virus (rAAV) mediated gene targeting. We achieved a 2x10-4 targeting frequency but only one-half of the targeted colonies harbored the SNP. Similarly, we succeeded in porcine LDLR gene knockout; however, targeted clones were often confounded by "bystander" cells with only random insertions of the targeting vector.;We turned to develop TALENs for efficient targeting of important genes. TALENs demonstrated high activity in both cultured primary fibroblasts and early stage embryos. A simple SB transposon based co-selection strategy enabled enrichment for TALEN modified cells and efficient isolation of modified clones: single gene mono- and bi-allelic modification was induced in up to 54% and 17% of colonies respectively. It also enabled isolation of colonies harboring large chromosomal deletions (10% of colonies) and inversions (4%) after treatment with two TALEN pairs. We derived miniature swine models of familial hypercholesterolemia from LDLR mono- and bi-allelic TALEN-knockout fibroblasts.;We next utilized TALEN and CRISPR/Cas9 stimulated homology-directed repair (HDR) to edit genes with oligonucleotide, plasmid, and rAAV templates without any drug selection. We first introgressed a bovine POLLED allele into horned dairy bull fibroblasts to circumvent manual dehorning. We also introduced single-nucleotide alterations or small indels into 14 additional genes in pig, cattle and sheep, into 10-50% of cells from fibroblast populations treated with TALEN mRNA and oligonucleotides. Up to 67% of propagated colonies harbored the intended edits and over one-half were homozygous. Some edits were naturally occurring SNP alleles, equivalent to non-meiotic inter- or intra-species introgression of valuable alleles. We created pig models for infertility and colon cancer from colonies with TALEN-HDR knockout alleles in DAZL and APC.