Large animal models are valuable tools for investigating human disease. Sheep, pigs, and goats often better recapitulate the anatomy and physiology of human organs and the complexity of human disease, thereby enhancing their clinical relevance compared to rodents. CRISPR-Cas9 and somatic cell nuclear transfer (SCNT) enable the generation of large animal models with greater precision, versatility, and genetic uniformity. The primary benefit of this approach, compared with zygote microinjection, is the ability to confirm in vitro whether the desired genetic modification and potential off-target mutations are present in gene-edited cells prior to animal production. Moreover, SCNT eliminates the chance of genetic mosaicism, which frequently results from zygote microinjection. Here, we describe the generation of gene-edited ovine cells through non-homologous end-joining (NHEJ) and homology-directed repair (HDR), followed by the production of cloned embryos carrying the mutations of interest. Genetic modifications are introduced by transfecting cultured somatic cells, typically fetal fibroblasts, with the CRISPR-Cas9 system. Mutation efficiency in pooled cells is assessed by polymerase chain reaction (PCR) and Sanger sequencing of edited genes and analyzed using Tracking of Indels by DEcomposition (TIDE)/Tracking of Insertions, Deletions, and Recombination events (TIDER) software. Limiting dilution of the pooled cells is performed to obtain single-cell-derived colonies, which are screened by PCR and DNA sequencing of edited genes. Donor cells with the edit(s) of interest are subsequently expanded and used for the generation of embryos by SCNT. After limiting dilution and cell screening, 22/114 (19.3%) of colonies modified through NHEJ contained knockout (KO) mutations and 4/56 (7.1%) of colonies modified with HDR contained the F508del mutation. A total of 370 genetically modified embryos were created from four colonies. These methods are successfully used for precise gene editing in fetal fibroblasts and generation of genetically engineered embryos to produce ovine models of human disease.
