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.
Highlights
Formation or survival of prospermatogonia leads to a loss of germline prior to establishment of the spermatogenic lineage but seminiferous tubules remain intact[10,11]
We have shown that direct injection of CRISPR reagents into the cytoplasm of in vivo and in vitro fertilized zygotes resulted in 18 piglets from three litters, with all piglets showing modifications in one (1 piglet) or both alleles (17 piglets)
The high incidence of mosaicism could be a result of injection into in vivo zygotes that are close to their first cell division resulting in independent editing events in two cells
Summary
Formation or survival of prospermatogonia leads to a loss of germline prior to establishment of the spermatogenic lineage but seminiferous tubules remain intact[10,11]. Nanos[2] knockout male mice are sterile due to apoptosis of prospermatogonia shortly after birth, homozygous knockout females, as well as heterozygous knockout males and females possess normal germline and are fertile[10,11]. The choice of NANOS2 for targeting in livestock has two advantages: (1) homozygous NANOS2 knockout males will be deficient in SSCs and will serve as ideal recipients for transplantation; and (2) heterozygous knockout males when bred to homozygous knockout females (both fertile) will efficiently propagate the line and generate NANOS2 null animals at high frequency. Males with heterozygous frameshift mutations and females with homozygous and heterozygous frameshift mutations are fertile These observations confirm that genetic modification of NANOS2 in pigs phenocopy mutations in mice
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