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

Authors: FLOREZ, JULIO - Acceligen Inc; MARTINS, KYRA - Acceligen Inc; SOLIN, STACI - Recombinetics, Inc; BOSTROM, JONATHAN - Acceligen Inc; RODRIGUEZ-VILLAMIL, PAULA - Acceligen Inc; ONGARATTO, FELIPE - Acceligen Inc; LARSON, SABREENA - Acceligen Inc; GANBAATAR, UYANGA - Recombinetics, Inc; COUTTS, ALEXANDER - Recombinetics, Inc; KERN, DOUG - Recombinetics, Inc; Murphy, Thomas - Tom; KIM, EUI-SOO - Acceligen Inc; CARLSON, DANIEL - Recombinetics, Inc; HUISMAN, ABE - Hendrix Genetics; SONSTEGARD, TAD - Acceligen Inc; Lents, Clay

Submitted to: Frontiers in Genetics
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/5/2022
Publication Date: 1/4/2023
Citation: Florez, J.M., Martins, K., Solin, S., Bostrom, J.R., Rodriguez-Villamil, P., Ongaratto, F., Larson, S.A., Ganbaatar, U., Coutts, A.W., Kern, D., Murphy, T.W., Kim, E., Carlson, D.F., Huisman, A., Sonstegard, T.S., Lents, C.A. 2023. CRISPR/Cas9-editing of KISS1 to generate pigs with hypogonadotropic hypogonadism as a castration free trait. Frontiers in Genetics. 13. Article 1078991. https://doi.org/10.3389/fgene.2022.1078991.
DOI: https://doi.org/10.3389/fgene.2022.1078991

Interpretive Summary: Male piglets destined for pork production are routinely castrated to prevent development of aggressive male behaviors that increase the risk of injures to other pigs and workers, and to meet consumer expectations for pork quality. Gene editing technologies provide opportunity to develop alternatives to castration. Researchers in collaboration with ARS scientists at Clay Center, Nebraska, edited a key gene in the reproductive system called KISS1. Scientists found that editing the KISS1 gene could keep testes of male pigs from developing, but all other aspects of pig development were normal. This indicates the potential for genomic based alternatives to castration and could lead to improved animal welfare.

Technical 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 prevent 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 of 5% to 97% and did not phenotypically differ from their wild-type counterparts. In contrast, 4 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 in KISS1 KO breeding stock to fully realize the potential of KISS1 gene edits to eliminate the need for surgical castration.