CRISPR/Cas9-editing of KISS1 to generate pigs with hypogonadotropic hypogonadism as a castration free trait

Abstract

Introduction: Most male pigs are surgically castrated to avoid puberty-derived boar taint and aggressiveness. However, this surgical intervention represents a welfare concern in swine production. Disrupting porcine KISS1 is hypothesized to delay or abolish puberty by inducing variable hypogonadotropism and thus preventing the need for castration. Methods: To test this hypothesis, we generated the first KISS1-edited large animal using CRISPR/Cas9-ribonucleoproteins and single-stranded donor oligonucleotides. The targeted region preceded the sequence encoding a conserved core motif of kisspeptin. Genome editors were intracytoplasmically injected into 684 swine zygotes and transferred to 19 hormonally synchronized surrogate sows. In nine litters, 49 American Yorkshire and 20 Duroc liveborn piglets were naturally farrowed. Results: Thirty-five of these pigs bore KISS1-disruptive alleles ranging in frequency from 5% to 97% and did not phenotypically differ from their wild-type counterparts. In contrast, four KISS1-edited pigs (two boars and two gilts) with disruptive allele frequencies of 96% and 100% demonstrated full hypogonadotropism, infantile reproductive tracts, and failed to reach sexual maturity. Change in body weight during development was unaffected by editing KISS1. Founder pigs partially carrying KISS1-disruptive alleles were bred resulting in a total of 53 KISS1 ^+/+, 60 KISS1 ^+/-, and 34 KISS1 ^-/- F1 liveborn piglets, confirming germline transmission. Discussion: Results demonstrate that a high proportion of KISS1 alleles in pigs must be disrupted before variation in gonadotropin secretion is observed, suggesting that even a small amount of kisspeptin ligand is sufficient to confer proper sexual development and puberty in pigs. Follow-on studies will evaluate fertility restoration in KISS1 KO breeding stock to fully realize the potential of KISS1 gene edits to eliminate the need for surgical castration.

Keywords: animal welfare; boar taint; embryo editing; homology-directed repair; kisspeptin; knockout; pig puberty.

FIGURE 1

Generation and genotyping of KISS1-edited pigs via zygote injection of CRISPR/Cas9 and single-stranded donor oligonucleotides (ssODNs). (A) Graphics depicting the genomic structure of S. scrofa KISS1, the gene sites bound by the designed sgRNA as well as the homology-directed repair (HDR) templates, and encoding the conserved amino acid core of this protein. At the lower part, it is shown the kisspeptin sequence alignment among different mammalian species in the region flanking the highly conserved core of this protein. (B) Schematic representation of CRISPR/Cas9 RNPs targeting KISS1 besides both ssODNs, and sequences of these HDR templates used to introduce a stop codon and the HindIII restriction site (HDR^SC) as well as synonymous SNPs, to change the PAM sequence, to reduce recutting and generate the AcuI restriction site (HDR^BM). (C) Schematic of the intracytoplasmic microinjection of CRISPR components and ssODNs into porcine zygotes, transfer of injected embryos, obtention of pigs, and their genotyping for the targeted locus. (D) Bar graph showing gene-edited pigs grouped according to their KISS1-disruptive editing percentages and depicting the allele names and percentages in each individual. In the X-axis are the pig IDs, in which F stands for female, M for male, Y for Yorkshire, D for Duroc, and SB for stillborn.

FIGURE 2

Reproductive organ phenotyping and growth-related traits of KISS1-edited pigs. (A) Representation of individual testicular volumes collected across different time points, where each panel corresponds to a KISS1-disruption group. “Cry” stands for cryptorchid. (B) In vivo and ex vivo comparison of representative WT and KISS1 KO testicles. (C) Comparison of the testicular weight of WT and KISS1-edited boars. (D,E) Comparison of representative WT and KISS1 KO gilt reproductive tracts (D) and ovaries (E). (F) Follicle count and measurements of ovaries. In (C) and (F), values are individually represented for pigs with KISS1-disruptive editing percentages above 90% and as means for the other groups. (G,H) Body weights (LSmeans ± SE; age, p < .001) (G) and average daily gain of weights (LS means ± SE; birth weight, p < .001) (H) across the evaluated time points (±4 days) and periods, respectively. (I) Scatter plot of total average daily gain of weight (i.e., from birth to 160 days of age) with lines indicating the KISS1 KO pigs. In (G–I), results are grouped by the KISS1-disruptive editing percent.

FIGURE 3

Immunohistochemical analysis of germ and Sertoli cell markers in seminiferous tubules from WT and KISS1 KO pigs. Boars were 8 months old. DAPI was applied as nuclear staining and is represented in blue fluorescence. Combined images of structure, targeted proteins, and nuclei are shown in the last column. Scale bar = 100 µm.

FIGURE 4

Hormone profiles of KISS1-edited pigs. (A–D) Serum concentration (LSmeans ± SE) in boars and gilts of FSH [(A) and (B), respectively] and LH [(C) and (D), respectively] during development by KISS1-disruption group. (E) Developmental change in serum concentrations (LS means ± SE) of testosterone in boars according to the KISS1-disruptive editing percent.

FIGURE 5

Confirmation of KISS1-edited allele transmission. (A) KISS1 allele frequencies of mosaic F0 boar candidates for breeding, calculated using DNA from tail tissue and spermatozoa. (B) Inherited KISS1 alleles by F1 piglets, and the number of genotyped pigs in each litter according to their sex-breed (first bar) or genotype category (second bar). “DEP” stands for disruptive editing percent and “Δ” means deletion.