Generation of NANOS2 mutant pigs

In pigs, NANOS2 is a single exonic gene with an open reading frame (ORF) of 417 bp. A candidate CRISPR/Cas9 single guide RNA (sgRNA) was designed to target 48 to 70 bp proximal to the translational start site (AUG) (Supplementary Figure S1). To produce pigs with mutations in the NANOS2 gene, donor animals were estrous synchronized, artificially inseminated, and in vivo fertilized zygotes and unfertilized oocytes were surgically recovered. The in vivo fertilized zygotes were microinjected with a mixture of sgRNAs and a SpCas9 RNA expression construct (Fig. 1a), and 30 zygotes were transferred into each of the synchronized recipient gilts. The remaining unfertilized oocytes were in vitro fertilized, injected with the CRISPR cocktail and all 54 zygotes surgically transferred. Three out of four recipients from the embryo transfers were confirmed pregnant that resulted in the birth of 12 males and 6 females, total 18 live piglets (Fig. 1b and Supplementary Figure S2). Genotyping of ear notch biopsies identified an array of mutations with all piglets showing either mono- or bi-allelic modifications and no wildtype animals (Fig. 1c and Supplementary Figure 2). Among the male offspring, piglets #137, 144, 146 and 251 showed bi-allelic frameshifting mutations, piglet #136 had three confirmed edited alleles (mosaic), whereas, piglets #142, 143, 147, and 148 contained at least one 3 bp in-frame deletion resulting in the loss of valine (at position 19). We hypothesized that 3 bp in-frame deletions would be functional, and piglets #142, 143, 147 and 148 were classified as heterozygous null, and piglets #137, 144, 146 and 251 with out-of-frame bi-allelic modifications as homozygous knockouts. Three of the littermates (2 heterozygous and 1 homozygous null) were euthanized prior to or around the time of weaning and were, therefore not included in the study.

Figure 1: Generation of NANOS2 gene edited pigs. (a) Schematics of Cas9:GFP expression vectors containing an HA tag, nuclear localization signal (NLS) and CMV promoter for mammalian in vivo expression (top) or T7 promoter for in vitro transcription (bottom). (b) Distribution of genotypes for 20 genome edited piglets of 3 litters produced from microinjected in vivo derived embryos (n = 18) and 2 somatic cell nuclear transfer (SCNT) piglets. (c) Genotype of 9 mono- and bi-allelically edited boars from ear biopsy and semen DNA samples. Two of the knockout boars that had ORF disrupting mutations and no spermatogenesis are highlighted in yellow. The alleles were determined by PCR amplification and sub-cloning of a 500 bp amplified fragment from each piglet (flanking the target site of NANOS2 genomic site) into PCR2.1 plasmid and sequencing of 10 bacterial clones from each piglet. Two of the knockout boars (#144 and #146) that had ORF disrupting mutations had no spermatogenesis, and hence no genotyping from spermatozoa could be performed. Boar #147 was euthanized and no further genotyping was performed. Full size image

Growth and development of NANOS2 mutant male pigs

We reasoned that if NANOS2 plays a role in the development and function of cell lineages other than the male germline in pigs, overall growth and development would be affected. To assess this, we measured the weight of male animals at periodic intervals from birth through adulthood (Fig. 2a). At birth, the average weight of homozygous knockout NANOS2 male pigs was 1.3 ± 0.2 kg (mean ± STDV and n = 4) which was not different (P = 0.19) compared to the average weight of heterozygous knockout males, 1.9 ± 0.3 kg (n = 4). Likewise, there was no difference (P = 0.42) in body weight between the homozygous and heterozygous groups at weaning (8.0 ± 1.1 vs. 9.1 ± 0.2, respectively; n = 4 of each genotype), or at 3, 6, 9, or 12 months of age (Fig. 2a). In addition, no gross differences in the appearance or behavior of animals were observed. Note that homozygous knockout pig #144 and heterozygous pig #147 became ill around 7 months of age and had to be euthanized, thus these animals are not included in the full analysis. Boar #144 developed lameness in the right forelimb that limited standing and movement and boar #147 developed an esophageal blockage that impaired his ability to eat. To assess testis development, we used ultrasound imaging to measure the diameter of each testis at periodic age points (Fig. 2b and c). At 3 months of age, the average paired testis diameter for homozygous null edited pigs was 26.1 ± 1.3 mm (mean ± SEM and n = 4 of each genotype) which was not different (P = 0.24) from the 23.7 ± 0.8 mm diameter for heterozygous animals (n = 4). At 8 months of age, the average paired testis diameter for homozygous null edited boars #137 and 251 was 61.9 ± 2.6 mm (mean ± STDV) and 51.9 ± 6.0 mm (mean ± STDV), respectively, which was similar to the 62.1 ± 4.5 mm average paired testis diameter of the heterozygous edited group. In contrast, the average paired testis diameter for homozygous edited boar #146 was reduced by ~40% to 37.5 ± 1.8 (mean ± STDV) compared to the heterozygous group. A phenotype of reduced testis size could be caused by disruption of the hypothalamic-pituitary-gonadal (HPG) axis. To assess this possibility, we measured serum testosterone concentration in serial samples (4 time points at 15 min intervals) from heterozygous and homozygous edited animals at 6 months of age (Fig. 2d). Although the mean value for each boar varied, all were within the normal range for an adult male15. Taken together, these findings suggested that loss of NANOS2 expression does not alter normal growth and development in male pigs. However, overall development of testes is impaired, at least in some knockout animals, possibly due to disrupted establishment of the germline. The disparity in testis diameter phenotype among the homozygous edited boars suggested variability in NANOS2 loss-of-function or mosaicism of the edited alleles in some animals.

Figure 2: Growth and development of NANOS2 gene edited male pigs. (a) Body weight of individual NANOS2 heterozygous edited (#142, 143, 147, 148) and homozygous edited (#137, 144, 146, 251) male pigs at periodic points in development from birth (0 months, MO) through adulthood (12 MO). Note that data are not presented for pigs #144 and 147 at the age points of 6-12 MO because they were euthanized around 7 MO of age for health reasons. (b) Representative ultrasound image of a testis from NANOS2 gene edited pigs. Image is from bi-allelic NANOS2 edited pig #137. (c) Testis diameter measurements from ultrasound imaging for individual NANOS2 mono-allelic edited (#142, 143, 147, 148) and bi-allelic edited (#137, 144, 146, 251) male pigs during prepubertal development (3 MO) and at puberty (6 MO). Note that data are not presented for pigs #144 and 147 at the 6 MO age point because of deteriorating health. (d) Serum testosterone concentration for individual NANOS2 mono-allelic edited (#142, 143, 148) and bi-allelic edited (#137, 146, 251) male pigs at 7 months of age. Data are presented as mean ± STDV for all pigs of each genotype. Note that in the figure ND stands for “Not determined” as pigs #144 and 147 were euthanized prior to the serial blood sampling. Full size image

Germline ablation of NANOS2 male knockout pigs

To assess the testis phenotype of NANOS2 mutant pigs further, we collected testicular biopsies from both homozygous knockout and heterozygous animals at the pubertal age of 6–8 months. Evaluation of cross-sections revealed the presence of intact seminiferous tubules for both genotypes and germ cells were clearly evident in tubules of all heterozygous null males (Fig. 3a), as well as homozygous edited boars #137 and 251 (Fig. 3b). However, no germ cells were apparent in tubules of homozygous edited boars #144 (Supplementary Figure S3) and 146 (Fig. 3b). In adulthood, semen samples were collected from the boars using a dummy apparatus. Samples from heterozygous edited animals contained numerous sperm with a progressive motility and normal morphology of >90% (Fig. 3a). Similarly, sperm were present in the ejaculates of homozygous edited boars #137 and 251 (Fig. 3b). In contrast, no sperm were detectable in samples from homozygous edited boar #146 (Fig. 3b). Note that boar #144 became ill and semen collection was not possible. Taken together, these results led us to speculate that bi-allelic edited boars #137 and 251 might be mosaics in which at least one functional NANOS2 allele was intact in the germline. Indeed, genotyping of semen from boars #251 and 137 revealed at least one non-inactivating NANOS2 allele (Fig. 1c). Thus, bi-allelically edited boars #144 and 146 were true NANOS2 knockouts; whereas, bi-allelically edited boars #137 and 251 were mosaics with a functional NANOS2 allele in germ cells. Collectively, these findings demonstrate that the frameshifting true knockout of NANOS2 results in germline specific ablation in males (boars #144 and 146), which is conserved from mouse to pig.

Figure 3: Testicular phenotype of NANOS2 gene edited male pigs. (a and b) Representative images of cross-sections from testicular parenchyma (upper panels) and ejaculates (lower panels) from NANOS2 mono-allelic (a) and bi-allelic (b) edited pigs at adulthood (6–8 months of age). Note that the cross-section of testicular tissue from bi-allelic knockout pig #146 lacks germline and the ejaculate is devoid of sperm. Full size image

Fertility of female NANOS2 mutant pigs

From the embryo transfers, we identified 6 female piglets, none of which possessed homozygous out-of-frame mutations (Supplementary Table S1). We therefore nucleofected somatic cells with CRIPSR/Cas9 GFP plasmid and a sgRNA expression vector targeting the NANOS2 locus. One of the clonal lines that was bi-allelic knockout for NANOS2 was used as a donor for somatic cell nuclear transfer (SCNT), which yielded true knockout female piglets (Fig. 4a). Note that nuclear transfer is performed with one starting cell of a known genotype as the nuclear donor. Therefore, unlike the animals derived from zygote injections, mosaicism or the presence of an unaccounted 3rd allele is not possible in SCNT -derived animals. Beginning at 8 months of age, the knockout females exhibited normal estrous cyclicity (19–21 days) and evaluation of cross-sections from the ovaries of one piglet (euthanized because of musculoskeletal issues) revealed intact follicles and normal folliculogenesis (Fig. 4b). Breeding of a clonal NANOS2 knockout littermate resulted in a successful pregnancy (confirmed by ultrasound at 4 weeks). Lastly, we utilized sperm from the mosaic heterozygous knockout boar #142 to breed NANOS2 mutant sows #145 and #149 (heterozygous knockouts), and boar #143 was bred to sow #140 (heterozygous knockout); all of which resulted in pregnancies and produced litters (Supplementary Table S2). Taken together, these findings demonstrate that the germline is intact in females that are deficient for NANOS2.