Comparative potato genome editing: Agrobacterium tumefaciens-mediated transformation and protoplasts transfection delivery of CRISPR/Cas9 components directed to StPPO2 gene

Autores
Gonzalez, Matías Nicolás; Massa, Gabriela Alejandra; Anderson, Mariette; Decima Oneto, Cecilia Andrea; Turesson, Helle; Olsson, Niklas; Fält, Ann Sofie; Hofvander, Per; Feingold, Sergio Enrique
Año de publicación
2021
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
Delivery of the CRISPR/Cas9 components to the plant cells is a key step in its application as a genome editing tool. Here, we compared Agrobacterium-mediated transformation and protoplast transfection with CRISPR/Cas9 components for potato genome editing. Two sgRNAs were designed to simultaneously direct Cas9 to the StPPO2 gene encoding for a tuber polyphenol oxidase (PPO). A binary vector (CR-PPO) was utilized for either Agrobacterium-mediated transformation or for transient expression in protoplasts, while ribonucleoprotein complexes (RNP-PPO) were additionally assayed in protoplasts. Editing efficiency varied, yielding 9.6%, 18.4% and 31.9% of edited lines from Agrobacterium-mediated transformation, RNP-PPO and CR-PPO transient expression in protoplasts, respectively. Furthermore, only the CR-PPO transient expression resulted in lines edited in all four StPPO2 alleles, observed in 46% of the edited lines and confirmed by tuber PPO activity and enzymatic browning analysis. Lines with on-target DNA insertions were found from all three approaches and characterized by sequencing. The dual-sgRNA strategy resulted in a low incidence of the targeted deletion, likely due to contrasting efficiencies between sgRNAs, that was partially evident in the in silico analysis. Our results demonstrate that gene editing efficiency in potato depends on the CRISPR/Cas9 delivery strategy and provide insights to consider when selecting the appropriate methodology.
EEA Balcarce
Fil: González, Matías Nicolás. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto de Innovación para la Producción Agropecuaria y el Desarrollo Sostenible; Argentina.
Fil: Massa, Gabriela Alejandra. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto de Innovación para la Producción Agropecuaria y el Desarrollo Sostenible; Argentina.
Fil: Massa, Gabriela Alejandra. Universidad de Buenos Aires. Facultad de Agronomía; Argentina.
Fil: Anderson, Mariette. Swedish University of Agricultural Sciences. Department of Plant Breeding; Suecia.
Fil: Décima Oneto, Cecilia Andrea. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto de Innovación para la Producción Agropecuaria y el Desarrollo Sostenible; Argentina.
Fil: Décima Oneto, Cecilia Andrea. Universidad de Buenos Aires. Facultad de Agronomía; Argentina.
Fil: Turesson, Helle. Swedish University of Agricultural Sciences. Department of Plant Breeding; Suecia.
Fil: Storani, Leonardo. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto de Innovación para la Producción Agropecuaria y el Desarrollo Sostenible; Argentina.
Fil: Olsson, Niklas. Swedish University of Agricultural Sciences. Department of Plant Breeding; Suecia.
Fil: Fält , Ann Sofie. Swedish University of Agricultural Sciences. Department of Plant Breeding; Suecia.
Fil: Hofvander, Per. Swedish University of Agricultural Sciences. Department of Plant Breeding; Suecia.
Fil: Feingold, Sergio Enrique. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto de Innovación para la Producción Agropecuaria y el Desarrollo Sostenible; Argentina.
Fuente
Plant Cell, Tissue and Organ Culture 145 : 291-305 (2021)
Materia
Papa
Edición de Genes
Protoplastos
Agrobacterium Tumefaciens
Catecol Oxidasa
Potatoes
Gene Editing
Protoplasts
Catechol Oxidase
Nivel de accesibilidad
acceso restringido
Condiciones de uso
Repositorio
INTA Digital (INTA)
Institución
Instituto Nacional de Tecnología Agropecuaria
OAI Identificador
oai:localhost:20.500.12123/9204
Acceder
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oai_identifier_str oai:localhost:20.500.12123/9204
network_acronym_str INTADig
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network_name_str INTA Digital (INTA)
spelling Comparative potato genome editing: Agrobacterium tumefaciens-mediated transformation and protoplasts transfection delivery of CRISPR/Cas9 components directed to StPPO2 geneGonzalez, Matías NicolásMassa, Gabriela AlejandraAnderson, MarietteDecima Oneto, Cecilia AndreaTuresson, HelleOlsson, NiklasFält, Ann SofieHofvander, PerFeingold, Sergio EnriquePapaEdición de GenesProtoplastosAgrobacterium TumefaciensCatecol OxidasaPotatoesGene EditingProtoplastsCatechol OxidaseDelivery of the CRISPR/Cas9 components to the plant cells is a key step in its application as a genome editing tool. Here, we compared Agrobacterium-mediated transformation and protoplast transfection with CRISPR/Cas9 components for potato genome editing. Two sgRNAs were designed to simultaneously direct Cas9 to the StPPO2 gene encoding for a tuber polyphenol oxidase (PPO). A binary vector (CR-PPO) was utilized for either Agrobacterium-mediated transformation or for transient expression in protoplasts, while ribonucleoprotein complexes (RNP-PPO) were additionally assayed in protoplasts. Editing efficiency varied, yielding 9.6%, 18.4% and 31.9% of edited lines from Agrobacterium-mediated transformation, RNP-PPO and CR-PPO transient expression in protoplasts, respectively. Furthermore, only the CR-PPO transient expression resulted in lines edited in all four StPPO2 alleles, observed in 46% of the edited lines and confirmed by tuber PPO activity and enzymatic browning analysis. Lines with on-target DNA insertions were found from all three approaches and characterized by sequencing. The dual-sgRNA strategy resulted in a low incidence of the targeted deletion, likely due to contrasting efficiencies between sgRNAs, that was partially evident in the in silico analysis. Our results demonstrate that gene editing efficiency in potato depends on the CRISPR/Cas9 delivery strategy and provide insights to consider when selecting the appropriate methodology.EEA BalcarceFil: González, Matías Nicolás. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto de Innovación para la Producción Agropecuaria y el Desarrollo Sostenible; Argentina.Fil: Massa, Gabriela Alejandra. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto de Innovación para la Producción Agropecuaria y el Desarrollo Sostenible; Argentina.Fil: Massa, Gabriela Alejandra. Universidad de Buenos Aires. Facultad de Agronomía; Argentina.Fil: Anderson, Mariette. Swedish University of Agricultural Sciences. Department of Plant Breeding; Suecia.Fil: Décima Oneto, Cecilia Andrea. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto de Innovación para la Producción Agropecuaria y el Desarrollo Sostenible; Argentina.Fil: Décima Oneto, Cecilia Andrea. Universidad de Buenos Aires. Facultad de Agronomía; Argentina.Fil: Turesson, Helle. Swedish University of Agricultural Sciences. Department of Plant Breeding; Suecia.Fil: Storani, Leonardo. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto de Innovación para la Producción Agropecuaria y el Desarrollo Sostenible; Argentina.Fil: Olsson, Niklas. Swedish University of Agricultural Sciences. Department of Plant Breeding; Suecia.Fil: Fält , Ann Sofie. Swedish University of Agricultural Sciences. Department of Plant Breeding; Suecia.Fil: Hofvander, Per. Swedish University of Agricultural Sciences. Department of Plant Breeding; Suecia.Fil: Feingold, Sergio Enrique. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto de Innovación para la Producción Agropecuaria y el Desarrollo Sostenible; Argentina.Springer Netherlands2021-04-28T10:45:48Z2021-04-28T10:45:48Z2021-01-25info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfhttp://hdl.handle.net/20.500.12123/9204https://link.springer.com/article/10.1007%2Fs11240-020-02008-91573-5044 (online)0167-6857 (print)https://doi.org/10.1007/s11240-020-02008-9Plant Cell, Tissue and Organ Culture 145 : 291-305 (2021)reponame:INTA Digital (INTA)instname:Instituto Nacional de Tecnología Agropecuariaenginfo:eu-repograntAgreement/INTA/PNBIO-1131024/AR./Desarrollo de sistemas alternativos de generación y utilización de variabilidad genética y su aplicación al mejoramiento de los cultivos.info:eu-repograntAgreement/INTA/2019-PE-E6-I115-001/2019-PE-E6-I115-001/AR./Edición génica, transgénesis y mutagénesis como generadores de nueva variabilidad en especies de interés agropecuarioinfo:eu-repo/semantics/restrictedAccess2025-09-04T09:48:52Zoai:localhost:20.500.12123/9204instacron:INTAInstitucionalhttp://repositorio.inta.gob.ar/Organismo científico-tecnológicoNo correspondehttp://repositorio.inta.gob.ar/oai/requesttripaldi.nicolas@inta.gob.arArgentinaNo correspondeNo correspondeNo correspondeopendoar:l2025-09-04 09:48:52.969INTA Digital (INTA) - Instituto Nacional de Tecnología Agropecuariafalse
dc.title.none.fl_str_mv Comparative potato genome editing: Agrobacterium tumefaciens-mediated transformation and protoplasts transfection delivery of CRISPR/Cas9 components directed to StPPO2 gene
title Comparative potato genome editing: Agrobacterium tumefaciens-mediated transformation and protoplasts transfection delivery of CRISPR/Cas9 components directed to StPPO2 gene
spellingShingle Comparative potato genome editing: Agrobacterium tumefaciens-mediated transformation and protoplasts transfection delivery of CRISPR/Cas9 components directed to StPPO2 gene
Gonzalez, Matías Nicolás
Papa
Edición de Genes
Protoplastos
Agrobacterium Tumefaciens
Catecol Oxidasa
Potatoes
Gene Editing
Protoplasts
Catechol Oxidase
title_short Comparative potato genome editing: Agrobacterium tumefaciens-mediated transformation and protoplasts transfection delivery of CRISPR/Cas9 components directed to StPPO2 gene
title_full Comparative potato genome editing: Agrobacterium tumefaciens-mediated transformation and protoplasts transfection delivery of CRISPR/Cas9 components directed to StPPO2 gene
title_fullStr Comparative potato genome editing: Agrobacterium tumefaciens-mediated transformation and protoplasts transfection delivery of CRISPR/Cas9 components directed to StPPO2 gene
title_full_unstemmed Comparative potato genome editing: Agrobacterium tumefaciens-mediated transformation and protoplasts transfection delivery of CRISPR/Cas9 components directed to StPPO2 gene
title_sort Comparative potato genome editing: Agrobacterium tumefaciens-mediated transformation and protoplasts transfection delivery of CRISPR/Cas9 components directed to StPPO2 gene
dc.creator.none.fl_str_mv Gonzalez, Matías Nicolás
Massa, Gabriela Alejandra
Anderson, Mariette
Decima Oneto, Cecilia Andrea
Turesson, Helle
Olsson, Niklas
Fält, Ann Sofie
Hofvander, Per
Feingold, Sergio Enrique
author Gonzalez, Matías Nicolás
author_facet Gonzalez, Matías Nicolás
Massa, Gabriela Alejandra
Anderson, Mariette
Decima Oneto, Cecilia Andrea
Turesson, Helle
Olsson, Niklas
Fält, Ann Sofie
Hofvander, Per
Feingold, Sergio Enrique
author_role author
author2 Massa, Gabriela Alejandra
Anderson, Mariette
Decima Oneto, Cecilia Andrea
Turesson, Helle
Olsson, Niklas
Fält, Ann Sofie
Hofvander, Per
Feingold, Sergio Enrique
author2_role author
author
author
author
author
author
author
author
dc.subject.none.fl_str_mv Papa
Edición de Genes
Protoplastos
Agrobacterium Tumefaciens
Catecol Oxidasa
Potatoes
Gene Editing
Protoplasts
Catechol Oxidase
topic Papa
Edición de Genes
Protoplastos
Agrobacterium Tumefaciens
Catecol Oxidasa
Potatoes
Gene Editing
Protoplasts
Catechol Oxidase
dc.description.none.fl_txt_mv Delivery of the CRISPR/Cas9 components to the plant cells is a key step in its application as a genome editing tool. Here, we compared Agrobacterium-mediated transformation and protoplast transfection with CRISPR/Cas9 components for potato genome editing. Two sgRNAs were designed to simultaneously direct Cas9 to the StPPO2 gene encoding for a tuber polyphenol oxidase (PPO). A binary vector (CR-PPO) was utilized for either Agrobacterium-mediated transformation or for transient expression in protoplasts, while ribonucleoprotein complexes (RNP-PPO) were additionally assayed in protoplasts. Editing efficiency varied, yielding 9.6%, 18.4% and 31.9% of edited lines from Agrobacterium-mediated transformation, RNP-PPO and CR-PPO transient expression in protoplasts, respectively. Furthermore, only the CR-PPO transient expression resulted in lines edited in all four StPPO2 alleles, observed in 46% of the edited lines and confirmed by tuber PPO activity and enzymatic browning analysis. Lines with on-target DNA insertions were found from all three approaches and characterized by sequencing. The dual-sgRNA strategy resulted in a low incidence of the targeted deletion, likely due to contrasting efficiencies between sgRNAs, that was partially evident in the in silico analysis. Our results demonstrate that gene editing efficiency in potato depends on the CRISPR/Cas9 delivery strategy and provide insights to consider when selecting the appropriate methodology.
EEA Balcarce
Fil: González, Matías Nicolás. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto de Innovación para la Producción Agropecuaria y el Desarrollo Sostenible; Argentina.
Fil: Massa, Gabriela Alejandra. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto de Innovación para la Producción Agropecuaria y el Desarrollo Sostenible; Argentina.
Fil: Massa, Gabriela Alejandra. Universidad de Buenos Aires. Facultad de Agronomía; Argentina.
Fil: Anderson, Mariette. Swedish University of Agricultural Sciences. Department of Plant Breeding; Suecia.
Fil: Décima Oneto, Cecilia Andrea. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto de Innovación para la Producción Agropecuaria y el Desarrollo Sostenible; Argentina.
Fil: Décima Oneto, Cecilia Andrea. Universidad de Buenos Aires. Facultad de Agronomía; Argentina.
Fil: Turesson, Helle. Swedish University of Agricultural Sciences. Department of Plant Breeding; Suecia.
Fil: Storani, Leonardo. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto de Innovación para la Producción Agropecuaria y el Desarrollo Sostenible; Argentina.
Fil: Olsson, Niklas. Swedish University of Agricultural Sciences. Department of Plant Breeding; Suecia.
Fil: Fält , Ann Sofie. Swedish University of Agricultural Sciences. Department of Plant Breeding; Suecia.
Fil: Hofvander, Per. Swedish University of Agricultural Sciences. Department of Plant Breeding; Suecia.
Fil: Feingold, Sergio Enrique. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto de Innovación para la Producción Agropecuaria y el Desarrollo Sostenible; Argentina.
description Delivery of the CRISPR/Cas9 components to the plant cells is a key step in its application as a genome editing tool. Here, we compared Agrobacterium-mediated transformation and protoplast transfection with CRISPR/Cas9 components for potato genome editing. Two sgRNAs were designed to simultaneously direct Cas9 to the StPPO2 gene encoding for a tuber polyphenol oxidase (PPO). A binary vector (CR-PPO) was utilized for either Agrobacterium-mediated transformation or for transient expression in protoplasts, while ribonucleoprotein complexes (RNP-PPO) were additionally assayed in protoplasts. Editing efficiency varied, yielding 9.6%, 18.4% and 31.9% of edited lines from Agrobacterium-mediated transformation, RNP-PPO and CR-PPO transient expression in protoplasts, respectively. Furthermore, only the CR-PPO transient expression resulted in lines edited in all four StPPO2 alleles, observed in 46% of the edited lines and confirmed by tuber PPO activity and enzymatic browning analysis. Lines with on-target DNA insertions were found from all three approaches and characterized by sequencing. The dual-sgRNA strategy resulted in a low incidence of the targeted deletion, likely due to contrasting efficiencies between sgRNAs, that was partially evident in the in silico analysis. Our results demonstrate that gene editing efficiency in potato depends on the CRISPR/Cas9 delivery strategy and provide insights to consider when selecting the appropriate methodology.
publishDate 2021
dc.date.none.fl_str_mv 2021-04-28T10:45:48Z
2021-04-28T10:45:48Z
2021-01-25
dc.type.none.fl_str_mv info:eu-repo/semantics/article
info:eu-repo/semantics/publishedVersion
http://purl.org/coar/resource_type/c_6501
info:ar-repo/semantics/articulo
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/20.500.12123/9204
https://link.springer.com/article/10.1007%2Fs11240-020-02008-9
1573-5044 (online)
0167-6857 (print)
https://doi.org/10.1007/s11240-020-02008-9
url http://hdl.handle.net/20.500.12123/9204
https://link.springer.com/article/10.1007%2Fs11240-020-02008-9
https://doi.org/10.1007/s11240-020-02008-9
identifier_str_mv 1573-5044 (online)
0167-6857 (print)
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repograntAgreement/INTA/PNBIO-1131024/AR./Desarrollo de sistemas alternativos de generación y utilización de variabilidad genética y su aplicación al mejoramiento de los cultivos.
info:eu-repograntAgreement/INTA/2019-PE-E6-I115-001/2019-PE-E6-I115-001/AR./Edición génica, transgénesis y mutagénesis como generadores de nueva variabilidad en especies de interés agropecuario
dc.rights.none.fl_str_mv info:eu-repo/semantics/restrictedAccess
eu_rights_str_mv restrictedAccess
dc.format.none.fl_str_mv application/pdf
dc.publisher.none.fl_str_mv Springer Netherlands
publisher.none.fl_str_mv Springer Netherlands
dc.source.none.fl_str_mv Plant Cell, Tissue and Organ Culture 145 : 291-305 (2021)
reponame:INTA Digital (INTA)
instname:Instituto Nacional de Tecnología Agropecuaria
reponame_str INTA Digital (INTA)
collection INTA Digital (INTA)
instname_str Instituto Nacional de Tecnología Agropecuaria
repository.name.fl_str_mv INTA Digital (INTA) - Instituto Nacional de Tecnología Agropecuaria
repository.mail.fl_str_mv tripaldi.nicolas@inta.gob.ar
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