Application of gene editing to livestock production

Brietta E Latham

Since the advent of animal agriculture, selective breeding has facilitated the genetic improvement of livestock for the continued provision of animal protein. Although assisted by modern reproductive technologies, such as artificial insemination, standard breeding methods are incapable of precision trait improvement. Through the use of gene editing, it is possible to not only specifically enhance traits related to growth and development but also impact animal health, welfare, and reproduction. Here, we demonstrate not only the range of uses for gene editing of livestock but also establish foundational knowledge for the development of new technologies to the application of these engineered genes in commercial settings. First, to facilitate the dissemination of high value genetics through natural mating, we established a strategy for the production and application of Surrogate Sires breeding technology in cattle. By CRISPR-Cas9 facilitated inactivation of NANOS2, a gene essential for the survival of precursor spermatogonia, sterile, germline-ablated males were produced to serve as recipients for donor germline transplantation. Engraftment of the transplanted spermatogonial stem cells resulted in the initiation of donor-derived spermatogenesis and attainment of fertility thereby demonstrating the ability of this approach to produce bulls with customizable sperm production. Furthermore, systematic breeding of a heterozygous NANOS2 knockout bull and NANOS2 edited heifers confirmed the concept of a NANOS2 knockout generator herd for production of future surrogate sires. In the second application of gene editing, strategies were developed and tested for the generation of short-tailed sheep. Although most sheep are born with long tails, these appendages are removed early in life to reduce the risk of a fly-borne disease called ‘fly strike’. To optimize animal welfare by preventing the complications and pain of tail docking while also limiting the risk of fly strike, we edited the gene TBXT which is known to be involved in tail formation. Upon knockout of the gene in mice, it was determined that although the total loss of Tbxt is lethal, tail truncation is a dominant trait in heterozygous animals. For implementation in sheep, CRISPR-Cas9 gene editing strategies were designed for the introduction of a two base pair swap in TBXT, a mutation found in naturally occurring short-tailed breeds. While the generation of short-tailed sheep using these strategies was attempted, successful conversion of the two base pairs was difficult to achieve. To overcome the lethality and inefficiency of the intended gene edit, methods for direct germline editing were established in mouse spermatogonial stem cells through electroporation. In the future, combination of this method with Surrogate Sires technology will allow for the distribution of engineered alleles, like the targeted TBXT mutation, without the complications of direct embryo editing. As the key component of both direct germline editing and surrogate sire transplantations, understanding undifferentiated spermatogonia is essential to the development of tools for the application of gene editing. Through the use of an experimentally tractable in vitro culture system, in which expression of the known biomarker Id4 is labeled with eGfp, live imaging was used to observe population dynamics including divisions and spontaneous fate changes that facilitated the maintenance of population composition. Challenging the undifferentiated spermatogonia through the alteration of the culture environment verified the essential elements of this system but also revealed an increase in the proportion of GFP+ SSCs on the verge of population collapse. In order to study the cellular mechanisms by which population composition is maintained, the undifferentiated spermatogonia population was modulated to contain only GFP- progenitor cells. Surprisingly, following a typical doubling period, the ability of these cells to maintain consistent population composition through the resurgence of the GFP+ SSC population, despite their commitment towards differentiation, was demonstrated. By expanding our understanding of SSC activity, we are better equipped to develop tools for the generation and application of gene edited livestock for the improvement of production efficiency.

Application of gene editing to livestock production

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