Sheep and Goat Genome Engineering: From Random Transgenesis to the CRISPR Era

Abstract

Sheep and goats are valuable livestock species that have been raised for their production of meat, milk, fiber, and other by-products. Due to their suitable size, short gestation period, and abundant secretion of milk, sheep and goats have become important model animals in agricultural, pharmaceutical, and biomedical research. Genome engineering has been widely applied to sheep and goat research. Pronuclear injection and somatic cell nuclear transfer represent the two primary procedures for the generation of genetically modified sheep and goats. Further assisted tools have emerged to enhance the efficiency of genetic modification and to simplify the generation of genetically modified founders. These tools include sperm-mediated gene transfer, viral vectors, RNA interference, recombinases, transposons, and endonucleases. Of these tools, the four classes of site-specific endonucleases (meganucleases, ZFNs, TALENs, and CRISPRs) have attracted wide attention due to their DNA double-strand break-inducing role, which enable desired DNA modifications based on the stimulation of native cellular DNA repair mechanisms. Currently, CRISPR systems dominate the field of genome editing. Gene-edited sheep and goats, generated using these tools, provide valuable models for investigations on gene functions, improving animal breeding, producing pharmaceuticals in milk, improving animal disease resistance, recapitulating human diseases, and providing hosts for the growth of human organs. In addition, more promising derivative tools of CRISPR systems have emerged such as base editors which enable the induction of single-base alterations without any requirements for homology-directed repair or DNA donor. These precise editors are helpful for revealing desirable phenotypes and correcting genetic diseases controlled by single bases. This review highlights the advances of genome engineering in sheep and goats over the past four decades with particular emphasis on the application of CRISPR/Cas9 systems.

Molecular mechanisms, off‐target activities, and clinical potentials of genome editing systems

“…CRISPR/Cas9 was suggested as a significant potential for clinical application, for example, in inherited genetic diseases, 61 infectious diseases, 62 cancer, 63 and so on. This system has been mostly applied for the creation of animal models for human diseases, 64 while there are few large animal models or clinical trials on cancer therapy via CRISPR/Cas‐mediated genome editing. T cells were modified by CRISPR/Cas9 to generate more potent chimeric antigen receptors T cells (CAR‐Ts), which was a prospective way to cure cancer 65 .…”

Large Animal Models of Huntington’s Disease: What We Have Learned and Where We Need to Go Next

“…The extant KI-HD-85Q and TgHD (N548) minipigs and transgenic (OVT73) sheep are certainly appropriate for early-stage PK-PD-safety and possibly biomarker testing, even without a fully manifest behavioral phenotype. CRISPR-mediated gene editing has been achieved in both minipigs [ 84 , 85 ] and sheep [ 83 , 86 ], paving the way for the generation of fully humanized (entire human m HTT genomic sequence) models and work is underway.…”

Selection for Favorable Health Traits: A Potential Approach to Cope with Diseases in Farm Animals

“…Genome editing is a powerful technology that can precisely modify the genome of an organism. The main genome editing tools are zinc-finger nucleases, transcription activator-like effector nucleases, and CRISPR/Cas9, which have been successfully employed to many farm animal species including swine, cattle, sheep, and poultry to cope with diseases at affordable costs by creating farm animals with disease-resistant genes [ 92 , 93 , 94 , 95 , 96 , 97 , 98 , 99 ]. There are clear opportunities especially in cases where conventional control options have shown limited success.…”