CRISPR/Cas9-editing of KISS1 to generate pigs with hypogonadotropic hypogonadism as a castration free trait
Autor(a) principal: | |
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Data de Publicação: | 2023 |
Outros Autores: | , , , , , , , , , , , , , , |
Tipo de documento: | Artigo |
Idioma: | eng |
Título da fonte: | Repositório Institucional da UNESP |
Texto Completo: | http://dx.doi.org/10.3389/fgene.2022.1078991 http://hdl.handle.net/11449/246674 |
Resumo: | 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. |
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CRISPR/Cas9-editing of KISS1 to generate pigs with hypogonadotropic hypogonadism as a castration free traitanimal welfareboar taintembryo editinghomology-directed repairkisspeptinknockoutpig pubertyIntroduction: 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.Foundation for Food and Agriculture ResearchAcceligen IncDepartment of Preventive Veterinary Medicine and Animal Reproduction School of Agricultural and Veterinarian Sciences São Paulo State University (Unesp)Recombinetics IncUSDA ARS U.S. Meat Animal Research CenterHypor Hendrix GeneticsDepartment of Preventive Veterinary Medicine and Animal Reproduction School of Agricultural and Veterinarian Sciences São Paulo State University (Unesp)Foundation for Food and Agriculture Research: 552176Acceligen IncUniversidade Estadual Paulista (UNESP)Recombinetics IncU.S. Meat Animal Research CenterHendrix GeneticsFlórez, Julio M. [UNESP]Martins, KyraSolin, StaciBostrom, Jonathan R.Rodríguez-Villamil, PaulaOngaratto, FelipeLarson, Sabreena A.Ganbaatar, UyangaCoutts, Alexander W.Kern, DougMurphy, Thomas W.Kim, Eui-SooCarlson, Daniel F.Huisman, AbeSonstegard, Tad S.Lents, Clay A.2023-07-29T12:47:27Z2023-07-29T12:47:27Z2023-01-04info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/articlehttp://dx.doi.org/10.3389/fgene.2022.1078991Frontiers in Genetics, v. 13.1664-8021http://hdl.handle.net/11449/24667410.3389/fgene.2022.10789912-s2.0-85146520624Scopusreponame:Repositório Institucional da UNESPinstname:Universidade Estadual Paulista (UNESP)instacron:UNESPengFrontiers in Geneticsinfo:eu-repo/semantics/openAccess2023-07-29T12:47:27Zoai:repositorio.unesp.br:11449/246674Repositório InstitucionalPUBhttp://repositorio.unesp.br/oai/requestopendoar:29462024-08-05T22:42:11.693856Repositório Institucional da UNESP - Universidade Estadual Paulista (UNESP)false |
dc.title.none.fl_str_mv |
CRISPR/Cas9-editing of KISS1 to generate pigs with hypogonadotropic hypogonadism as a castration free trait |
title |
CRISPR/Cas9-editing of KISS1 to generate pigs with hypogonadotropic hypogonadism as a castration free trait |
spellingShingle |
CRISPR/Cas9-editing of KISS1 to generate pigs with hypogonadotropic hypogonadism as a castration free trait Flórez, Julio M. [UNESP] animal welfare boar taint embryo editing homology-directed repair kisspeptin knockout pig puberty |
title_short |
CRISPR/Cas9-editing of KISS1 to generate pigs with hypogonadotropic hypogonadism as a castration free trait |
title_full |
CRISPR/Cas9-editing of KISS1 to generate pigs with hypogonadotropic hypogonadism as a castration free trait |
title_fullStr |
CRISPR/Cas9-editing of KISS1 to generate pigs with hypogonadotropic hypogonadism as a castration free trait |
title_full_unstemmed |
CRISPR/Cas9-editing of KISS1 to generate pigs with hypogonadotropic hypogonadism as a castration free trait |
title_sort |
CRISPR/Cas9-editing of KISS1 to generate pigs with hypogonadotropic hypogonadism as a castration free trait |
author |
Flórez, Julio M. [UNESP] |
author_facet |
Flórez, Julio M. [UNESP] Martins, Kyra Solin, Staci Bostrom, Jonathan R. Rodríguez-Villamil, Paula Ongaratto, Felipe Larson, Sabreena A. Ganbaatar, Uyanga Coutts, Alexander W. Kern, Doug Murphy, Thomas W. Kim, Eui-Soo Carlson, Daniel F. Huisman, Abe Sonstegard, Tad S. Lents, Clay A. |
author_role |
author |
author2 |
Martins, Kyra Solin, Staci Bostrom, Jonathan R. Rodríguez-Villamil, Paula Ongaratto, Felipe Larson, Sabreena A. Ganbaatar, Uyanga Coutts, Alexander W. Kern, Doug Murphy, Thomas W. Kim, Eui-Soo Carlson, Daniel F. Huisman, Abe Sonstegard, Tad S. Lents, Clay A. |
author2_role |
author author author author author author author author author author author author author author author |
dc.contributor.none.fl_str_mv |
Acceligen Inc Universidade Estadual Paulista (UNESP) Recombinetics Inc U.S. Meat Animal Research Center Hendrix Genetics |
dc.contributor.author.fl_str_mv |
Flórez, Julio M. [UNESP] Martins, Kyra Solin, Staci Bostrom, Jonathan R. Rodríguez-Villamil, Paula Ongaratto, Felipe Larson, Sabreena A. Ganbaatar, Uyanga Coutts, Alexander W. Kern, Doug Murphy, Thomas W. Kim, Eui-Soo Carlson, Daniel F. Huisman, Abe Sonstegard, Tad S. Lents, Clay A. |
dc.subject.por.fl_str_mv |
animal welfare boar taint embryo editing homology-directed repair kisspeptin knockout pig puberty |
topic |
animal welfare boar taint embryo editing homology-directed repair kisspeptin knockout pig puberty |
description |
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. |
publishDate |
2023 |
dc.date.none.fl_str_mv |
2023-07-29T12:47:27Z 2023-07-29T12:47:27Z 2023-01-04 |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/article |
format |
article |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
http://dx.doi.org/10.3389/fgene.2022.1078991 Frontiers in Genetics, v. 13. 1664-8021 http://hdl.handle.net/11449/246674 10.3389/fgene.2022.1078991 2-s2.0-85146520624 |
url |
http://dx.doi.org/10.3389/fgene.2022.1078991 http://hdl.handle.net/11449/246674 |
identifier_str_mv |
Frontiers in Genetics, v. 13. 1664-8021 10.3389/fgene.2022.1078991 2-s2.0-85146520624 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.none.fl_str_mv |
Frontiers in Genetics |
dc.rights.driver.fl_str_mv |
info:eu-repo/semantics/openAccess |
eu_rights_str_mv |
openAccess |
dc.source.none.fl_str_mv |
Scopus reponame:Repositório Institucional da UNESP instname:Universidade Estadual Paulista (UNESP) instacron:UNESP |
instname_str |
Universidade Estadual Paulista (UNESP) |
instacron_str |
UNESP |
institution |
UNESP |
reponame_str |
Repositório Institucional da UNESP |
collection |
Repositório Institucional da UNESP |
repository.name.fl_str_mv |
Repositório Institucional da UNESP - Universidade Estadual Paulista (UNESP) |
repository.mail.fl_str_mv |
|
_version_ |
1808129453010714624 |