Germline ablation achieved via CRISPR/Cas9 targeting of NANOS3 in bovine zygotes

Abstract

NANOS3 is expressed in migrating primordial germ cells (PGCs) to protect them from apoptosis, and it is known to be a critical factor for germline development of both sexes in several organisms. However, to date, live NANOS3 knockout (KO) cattle have not been reported, and the specific role of NANOS3 in male cattle, or bulls, remains unexplored. This study generated NANOS3 KO cattle via cytoplasmic microinjection of the CRISPR/Cas9 system in vitro produced bovine zygotes and evaluated the effect of NANOS3 elimination on bovine germline development, from fetal development through reproductive age. The co-injection of two selected guide RNA (gRNA)/Cas9 ribonucleoprotein complexes (i.e., dual gRNA approach) at 6 h post fertilization achieved a high NANOS3 KO rate in developing embryos. Subsequent embryo transfers resulted in a 31% (n = 8/26) pregnancy rate. A 75% (n = 6/8) total KO rate (i.e., 100% of alleles present contained complete loss-of-function mutations) was achieved with the dual gRNA editing approach. In NANOS3 KO fetal testes, PGCs were found to be completely eliminated by 41-day of fetal age. Importantly, despite the absence of germ cells, seminiferous tubule development was not impaired in NANOS3 KO bovine testes during fetal, perinatal, and adult stages. Moreover, a live, NANOS3 KO, germline-ablated bull was produced and at sexual maturity he exhibited normal libido, an anatomically normal reproductive tract, and intact somatic gonadal development and structure. Additionally, a live, NANOS3 KO, germline-ablated heifer was produced. However, it was evident that the absence of germ cells in NANOS3 KO cattle compromised the normalcy of ovarian development to a greater extent than it did testes development. The meat composition of NANOS3 KO cattle was unremarkable. Overall, this study demonstrated that the absence of NANOS3 in cattle leads to the specific deficiency of both male and female germ cells, suggesting the potential of NANOS3 KO cattle to act as hosts for donor-derived exogenous germ cell production in both sexes. These findings contribute to the understanding of NANOS3 function in cattle and have valuable implications for the development of novel breeding technologies using germline complementation in NANOS3 KO germline-ablated hosts.

Keywords: CRISPR/Cas9; NANOS3; bovine; embryo; gene editing; germline ablation.

FIGURE 1

Bovine NANOS3 targeting and PCR analysis strategy. Diagram of bovine NANOS3 showing the genomic locations of (A) long-range PCR primers (NANOS3_6 kb_2F, NANOS3_6 kb_2R), (B) short-range PCR primers (NANOS3_F2, NANOS3_R2), and (C) selected dual guides, sgRNA4 and sgRNA7 (dual gRNA_4 + 7) in relation to the highly conserved N-terminal (blue dashed line) and zinc finger (blue dotted line) domains. (D) Sanger sequencing results showing representative frameshift mutations from sgRNA4 (left) and sgRNA7 (right), and (E) Sanger sequencing results showing a targeted dual gRNA_4 + 7 297 bp deletion.

FIGURE 2

Collection of NANOS3 targeted bovine samples. (A) Images of 41d fetal urogenital ridges. (B) Comparison of size and average weight (bars) of testes at 90d of fetal age from NANOS3 KO (n = 4) versus control (n = 4) fetuses. (B) Comparison of size and average weight (bars) of testes at 283d of fetal age from NANOS3 KO (n = 2) versus control (n = 4) perinates. In both panel (B, C), testes from the same bovine sample (i.e., a testis pair) are indicated by the same shape and color. (D–I) Images of live NANOS3 edited cattle at 1-week-old (D, F, H) and 15-months-old (E, G, I). The top row (D, E) is heifer #854, middle row (F, G) is bull #838, and bottom row (H, I) is bull #3964. Scale bars are 1 cm.

FIGURE 3

Genotypic analysis of CRISPR/Cas9 NANOS3-presumptively-edited bovine samples (n = 8). (A) NANOS3 PCR results using short-range primers (NANOS3_2F, NANOS3_2R). (B) NANOS3 PCR results using long-range primers (NANOS3_6 kb_2F, NANOS3_6 kb_2R). Genetic wildtype (WT; +) band sizes are 610 bp (A) and 6,274 bp (B). (C) Diagram showing the Sanger sequencing results of small, in-frame mutations present in one of the edited NANOS3 alleles carried by 15 months_3964. There was a single bp substitution (C to T) and a 3 bp deletion near the sgRNA4 cut site and a 6 bp deletion near the sgRNA7 cut site. (D) Comparison of the WT bovine NANOS3 exon one amino acid sequence to 15 months_3964’s predicted amino acid sequence. The amino acid substitution is highlighted in yellow and italicized (P to L). The three deleted amino acids are represented by red font (WT) and dashes (15months_3964). The highly conserved N-terminal (dashed underline) and zinc finger binding (dotted underline) domains are underlined.

FIGURE 4

Germ cell-deficient phenotype in NANOS3 KO fetal and perinatal testes. Representative images of immunostaining for well-conserved germ cell markers. (A) Immunostaining for OCT4 (magenta) and PRDM1 (cyan) in the genital ridges of samples at 41 days of fetal age. (B) Immunostaining for DDX4 (cyan) in the testes of samples at 90 days and 283 days of fetal age. All sections were co-stained for DNA (Hoechst 33,342; gray). Scale bars are 100 μM. NANOS3 genotypes are noted in parentheses next to the sample name. Wildtype (WT): 100% WT, or non-mutated, genome. Knockout (KO): all alleles present in the sample were predicted to KO, or inactivate, NANOS3 (see Figure 3; Table 3).

FIGURE 5

scRNA-Seq analysis of 90 days fetal testes, comparing NANOS3 KO samples (n = 2) to control (ctrl) samples (n = 2). (A) UMAP plot of different cell populations of the fetal testis. Clusters were identified based on expression of known marker genes. (B) UMAP plot colored by individual samples (n = 4). (C) UMAP plot colored by treatment showing that only control samples are present in the primordial germ cell (PGC) cluster. (D) UMAPs showing differential expression of known late PGC/gonocyte markers, DAZL and DDX4.

FIGURE 6

scRNA-Seq differential gene expression analysis of NANOS3 KO and WT control (CT) testes at 90 days (top panel) and 283 days (bottom panel) of fetal age. Known-marker genes for different testicular somatic cell populations are listed across the bottom of the dot plots. Some genes are shared makers for 2 cell populations. The dot size represents the percent of cells present in a particular cell type cluster expressing a particular gene (larger dots indicate a greater proportion of cells). The color of the dot represents the average scaled expression level of a particular gene in a particular cell type cluster (darker indicates higher average expression). Conserved genes (black boxes) were defined as those genes that were more highly expressed (log-transformed fold-change ≥0.5) in a specific cell type of both treatments when compared to all other cell types at that timepoint. Differentially expressed genes (DEG; black asterisks) had significantly (p ≤ 0.05) different expression (log-transformed fold-change ≥0.5) between treatments. Some genes were both conserved and DEG. Abbreviations: Endothelial (Endo.), Peritubular Myoid (PTM), treatment (Trt), Hematopoietic Progenitor Cell Antigen CD34 (CD34), Von Willebrand Factor (VWF), Actin Alpha 2 Smooth Muscle (ACTA2), Transgelin (TAGLN), Insulin Like Growth Factor 2 (IGF2), Aristaless Related Homeobox (ARX), LIM Homeobox 9 (LHX9), Insulin Like Growth Factor Binding Protein 3 (IGFBP3), GATA Binding Protein 4 (GATA4), Inhibin Subunit Beta A (INHBA), Anti-Müllerian Hormone (AMH), and Inhibin Subunit Alpha (INHA).

FIGURE 7

scRNA-Seq analysis of 283 days perinatal testes, comparing a NANOS3 KO sample to a control (Ctrl) sample. (A) UMAP plot of different cell populations of the perinatal testis. Clusters were identified based on expression of known marker genes. (B) UMAP showing differential expression of known gonocyte marker DDX4. (C, D) UMAP plots colored by cell type for Ctrl (C) and KO (D) cells, showing the lack of gonocytes (i.e., absence of red dots in the black circle) in the NANOS3 KO sample.

FIGURE 8

Monthly body weight (left axis), scrotal circumference (right axis) (A), and reproductive hormone levels (B–F) of CRISPR/Cas9 NANOS3 edited cattle from 1 to 15 months old. Monthly serum hormone levels for male primary hormones, testosterone (ng/mL), AMH (ng/mL), and inhibin-B (pg/mL), and female primary hormones, estradiol (pg/mL) and progesterone (ng/mL). Sample key: NANOS3 KO heifer #854 (pink round dots), NANOS3 KO bull #838 (blue square dashes and blue bar for SC), NANOS3 edited bull #3964 (grey dashes and grey bar for SC), genetic wildtype (WT) control (ctrl) bull (black solid line).

FIGURE 9

Comparison of CRISPR/Cas9 NANOS3 KO bull #838 (A, C) and edited bull #3964s (B, D) reproductive tracts (A, B) and testes size (C, D) at 15 months of age. Scale bars are 5 cm. Ruler is 31.5 cm.

FIGURE 10

Histological analysis of CRISPR/Cas9 NANOS3 KO bull #838 and edited bull #3964s testes. (A) Representative images of DDX4 immunostained (cyan) testis cross-sections from KO bull #838 compared to an age matched, wildtype (WT) bull. All immunostained sections were co-stained for DNA (Hoechst 33,342; gray). (B) Representative images of H&E-stained testis cross-sections from KO bull #838, edited bull #3964, and an age matched, WT bull. Scale bars are 100 μM. Histology indicates that all samples have Sertoli cells lining the seminiferous tubules, but KO bull #838 lacks any spermatogenesis.

FIGURE 11

Phenotypic analysis of CRISPR/Cas9 NANOS3 KO Heifer #854 at 15 months of age. (A) Heifer #854s reproductive tract. (B, C) Heifer #854s left ovary (B) and right putative primitive streak (C). (D–J) Representative images of H&E-stained ovary cross-sections from heifer #854 showing a complete lack of oogenesis.