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.Methods: 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.

Horizon scanning of potential environmental applications of terrestrial animals, fish, algae and microorganisms produced by genetic modification, including the use of new genomic techniques

“…Targeted traits within the field of animal welfare are: the generation of GM hornless cattle, in order to avoid both painful dehorning and injuries in densely stocked barn environments ( Carlson et al, 2016 ), thermoregulation in cattle ( Hansen, 2020 ) or cold tolerance in pigs ( Zheng et al, 2017 ), and the delay of adolescence in pigs as an alternative to castration ( Flórez et al, 2022 ). However, it is controversial whether these applications can actually increase welfare ( Eriksson et al, 2018 ; De Graeff et al, 2019 ).…”

“…EFSA's scientific opinion on new developments in biotechnology applied to microorganisms, as mandated by the EC (2022), is expected to be published in June 2024 (Kagkli, 2023). Additional EC-mandated work by EFSA is under way on a scientific opinion on new developments in biotechnology applied to animals, a draft of which is due in January 2025 (Ardizzone, 2023).…”

Male animal sterilization: history, current practices, and potential methods for replacing castration

“…From this original effort, Hendrix Genetics announced an alliance with Recombinetics/Acceligen to further the development of this genetic approach to replace surgical castration ( 551 ). Using CRISPR/Cas9 technology, the alliance has also targeted KISS1 gene rather than the receptor and found a similar inhibition of puberty in the pig ( 552 ). However, this technology inevitably carries a problem, as homozygous sows and boars are sterile and unable to produce litters.…”