Generation of germline ablated male pigs by CRISPR/Cas9 editing of the NANOS2 gene

Abstract

Genome editing tools have revolutionized the generation of genetically modified animals including livestock. In particular, the domestic pig is a proven model of human physiology and an agriculturally important species. In this study, we utilized the CRISPR/Cas9 system to edit the NANOS2 gene in pig embryos to generate offspring with mono-allelic and bi-allelic mutations. We found that NANOS2 knockout pigs phenocopy knockout mice with male specific germline ablation but other aspects of testicular development are normal. Moreover, male pigs with one intact NANOS2 allele and female knockout pigs are fertile. From an agriculture perspective, NANOS2 knockout male pigs are expected to serve as an ideal surrogate for transplantation of donor spermatogonial stem cells to expand the availability of gametes from genetically desirable sires.

CRISPR in livestock: From editing to printing

“…This example represents a potential approach for reducing physical dehorning in dairy cattle without loss of productivity. As mentioned, the use of CRISPR to produce germline ablated male pigs has been proposed [50], and even thought this remains to be proven, it would offer new opportunities to finish with surgical castration in pigs [60,61]. Avoiding these practices in animal husbandry may encourage public support of genome-edited animals for food chain production.…”

TALEN‐mediated gene targeting in porcine spermatogonia

“…Also, while depletion of endogenous SSCs is not absolutely required for germ cell transplantation into immature testis of large animals such as goats, pigs, sheep, and cattle (Herrid, Vignarajan, Davey, Dobrinski, & Hill, 2006;Rodriguez-Sosa et al, 2009;Zeng et al, 2013), genetically modified pigs that lack endogenous germ cells have recently been reported (Park et al, 2017;Tan et al, 2013). As in vitro expansion of genetically modified porcine germ cells is currently not possible, these animals are expected to serve as efficient recipient models for transplantation of primary, geneedited donor cells.…”

“…A locus‐specific transgene knock‐in pig model has also been generated by using CRISPR/Cas‐9 and SCNT (Ruan et al, ). As a result of their high efficiency in mutagenesis, microinjection of TALENs, ZFNs, and CRISPRs/Cas‐9 into pig zygotes resulted in production of live piglets with engineered mutations (Hai, Teng, Guo, Li, & Zhou, ; Lillico et al, ; Park et al, ; Wang et al, ). However, CRISPR/Cas9 mediated gene editing in zygotes can result in target allele mosaicism in animals due to independent multiple gene editing events at early embryonic cleavage stages (Niu et al, ; Yen et al, ).…”

A strategy to exploit surrogate sire technology in livestock breeding programs

“…In this study, we performed simulations to develop a strategy for exploiting surrogate sire technology [1] in animal breeding programs. Surrogate sire technology allows the creation of males that lack their own germline cells, but have transplanted spermatogonial stem cells from other donor males.…”

“…This lag can also be represented with the number of years of genetic gain [7], e.g., ~4 years in a pig breeding program. Surrogate sire technology would allow a single elite nucleus male to give rise to very large numbers of commercial animals, by donating spermatogonial stem cells to its commercial surrogates [1]. This could shorten the lag between the nucleus, multiplication, and commercial layers.…”