Genome editing in potato via CRISPR-Cas9 ribonucleoprotein delivery

Autores
Andersson, Mariette; Turesson, Helle; Olsson, Niklas; Fält, Ann Sofie; Ohlsson, Pia; Gonzalez, Matías Nicolás; Samuelsson, Mathias; Hofvander, Per
Año de publicación
2018
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
Clustered regularly interspaced short palindromic repeats and CRISPR-associated protein-9 (CRISPR-Cas9) can be used as an efficient tool for genome editing in potato (Solanum tuberosum). From both a scientific and a regulatory perspective, it is beneficial if integration of DNA in the potato genome is avoided. We have implemented a DNA-free genome editing method, using delivery of CRISPR-Cas9 ribonucleoproteins (RNPs) to potato protoplasts, by targeting the gene encoding a granule bound starch synthase (GBSS, EC 2.4.1.242). The RNP method was directly implemented using previously developed protoplast isolation, transfection and regeneration protocols without further adjustments. Cas9 protein was preassembled with RNA produced either synthetically or by in vitro transcription. RNP with synthetically produced RNA (cr-RNP) induced mutations, i.e. indels, at a frequency of up to 9%, with all mutated lines being transgene-free. A mutagenesis frequency of 25% of all regenerated shoots was found when using RNP with in vitro transcriptionally produced RNA (IVT–RNP). However, more than 80% of the shoots with confirmed mutations had unintended inserts in the cut site, which was in the same range as when using DNA delivery. The inserts originated both from DNA template remnants from the in vitro transcription, and from chromosomal potato DNA. In 2–3% of the regenerated shoots from the RNP-experiments, mutations were induced in all four alleles resulting in a complete knockout of the GBSS enzyme function.
Fil: Andersson, Mariette. Swedish University Of Agricultural Sciences; Suecia
Fil: Turesson, Helle. Swedish University Of Agricultural Sciences; Suecia
Fil: Olsson, Niklas. Swedish University Of Agricultural Sciences; Suecia
Fil: Fält, Ann Sofie. Swedish University Of Agricultural Sciences; Suecia
Fil: Ohlsson, Pia. Swedish University Of Agricultural Sciences; Suecia
Fil: Gonzalez, Matías Nicolás. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires Sur. Estación Experimental Agropecuaria Balcarce; Argentina
Fil: Samuelsson, Mathias. Lyckeby Starch AB; Suecia
Fil: Hofvander, Per. Swedish University Of Agricultural Sciences; Suecia
Materia
POTATO
STARCH
TRANSGENE FREE
CRISPR CAS9
PROTOPLASTS
Nivel de accesibilidad
acceso abierto
Condiciones de uso
https://creativecommons.org/licenses/by/2.5/ar/
Repositorio
CONICET Digital (CONICET)
Institución
Consejo Nacional de Investigaciones Científicas y Técnicas
OAI Identificador
oai:ri.conicet.gov.ar:11336/100759
Acceder
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oai_identifier_str oai:ri.conicet.gov.ar:11336/100759
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling Genome editing in potato via CRISPR-Cas9 ribonucleoprotein deliveryAndersson, MarietteTuresson, HelleOlsson, NiklasFält, Ann SofieOhlsson, PiaGonzalez, Matías NicolásSamuelsson, MathiasHofvander, PerPOTATOSTARCHTRANSGENE FREECRISPR CAS9PROTOPLASTShttps://purl.org/becyt/ford/1.6https://purl.org/becyt/ford/1Clustered regularly interspaced short palindromic repeats and CRISPR-associated protein-9 (CRISPR-Cas9) can be used as an efficient tool for genome editing in potato (Solanum tuberosum). From both a scientific and a regulatory perspective, it is beneficial if integration of DNA in the potato genome is avoided. We have implemented a DNA-free genome editing method, using delivery of CRISPR-Cas9 ribonucleoproteins (RNPs) to potato protoplasts, by targeting the gene encoding a granule bound starch synthase (GBSS, EC 2.4.1.242). The RNP method was directly implemented using previously developed protoplast isolation, transfection and regeneration protocols without further adjustments. Cas9 protein was preassembled with RNA produced either synthetically or by in vitro transcription. RNP with synthetically produced RNA (cr-RNP) induced mutations, i.e. indels, at a frequency of up to 9%, with all mutated lines being transgene-free. A mutagenesis frequency of 25% of all regenerated shoots was found when using RNP with in vitro transcriptionally produced RNA (IVT–RNP). However, more than 80% of the shoots with confirmed mutations had unintended inserts in the cut site, which was in the same range as when using DNA delivery. The inserts originated both from DNA template remnants from the in vitro transcription, and from chromosomal potato DNA. In 2–3% of the regenerated shoots from the RNP-experiments, mutations were induced in all four alleles resulting in a complete knockout of the GBSS enzyme function.Fil: Andersson, Mariette. Swedish University Of Agricultural Sciences; SueciaFil: Turesson, Helle. Swedish University Of Agricultural Sciences; SueciaFil: Olsson, Niklas. Swedish University Of Agricultural Sciences; SueciaFil: Fält, Ann Sofie. Swedish University Of Agricultural Sciences; SueciaFil: Ohlsson, Pia. Swedish University Of Agricultural Sciences; SueciaFil: Gonzalez, Matías Nicolás. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires Sur. Estación Experimental Agropecuaria Balcarce; ArgentinaFil: Samuelsson, Mathias. Lyckeby Starch AB; SueciaFil: Hofvander, Per. Swedish University Of Agricultural Sciences; SueciaWiley Blackwell Publishing, Inc2018-12info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/100759Andersson, Mariette; Turesson, Helle; Olsson, Niklas; Fält, Ann Sofie; Ohlsson, Pia; et al.; Genome editing in potato via CRISPR-Cas9 ribonucleoprotein delivery; Wiley Blackwell Publishing, Inc; Physiologia Plantarum; 164; 4; 12-2018; 378-3840031-9317CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/url/http://doi.wiley.com/10.1111/ppl.12731info:eu-repo/semantics/altIdentifier/doi/10.1111/ppl.12731info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-09-03T09:45:18Zoai:ri.conicet.gov.ar:11336/100759instacron:CONICETInstitucionalhttp://ri.conicet.gov.ar/Organismo científico-tecnológicoNo correspondehttp://ri.conicet.gov.ar/oai/requestdasensio@conicet.gov.ar; lcarlino@conicet.gov.arArgentinaNo correspondeNo correspondeNo correspondeopendoar:34982025-09-03 09:45:18.548CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv Genome editing in potato via CRISPR-Cas9 ribonucleoprotein delivery
title Genome editing in potato via CRISPR-Cas9 ribonucleoprotein delivery
spellingShingle Genome editing in potato via CRISPR-Cas9 ribonucleoprotein delivery
Andersson, Mariette
POTATO
STARCH
TRANSGENE FREE
CRISPR CAS9
PROTOPLASTS
title_short Genome editing in potato via CRISPR-Cas9 ribonucleoprotein delivery
title_full Genome editing in potato via CRISPR-Cas9 ribonucleoprotein delivery
title_fullStr Genome editing in potato via CRISPR-Cas9 ribonucleoprotein delivery
title_full_unstemmed Genome editing in potato via CRISPR-Cas9 ribonucleoprotein delivery
title_sort Genome editing in potato via CRISPR-Cas9 ribonucleoprotein delivery
dc.creator.none.fl_str_mv Andersson, Mariette
Turesson, Helle
Olsson, Niklas
Fält, Ann Sofie
Ohlsson, Pia
Gonzalez, Matías Nicolás
Samuelsson, Mathias
Hofvander, Per
author Andersson, Mariette
author_facet Andersson, Mariette
Turesson, Helle
Olsson, Niklas
Fält, Ann Sofie
Ohlsson, Pia
Gonzalez, Matías Nicolás
Samuelsson, Mathias
Hofvander, Per
author_role author
author2 Turesson, Helle
Olsson, Niklas
Fält, Ann Sofie
Ohlsson, Pia
Gonzalez, Matías Nicolás
Samuelsson, Mathias
Hofvander, Per
author2_role author
author
author
author
author
author
author
dc.subject.none.fl_str_mv POTATO
STARCH
TRANSGENE FREE
CRISPR CAS9
PROTOPLASTS
topic POTATO
STARCH
TRANSGENE FREE
CRISPR CAS9
PROTOPLASTS
purl_subject.fl_str_mv https://purl.org/becyt/ford/1.6
https://purl.org/becyt/ford/1
dc.description.none.fl_txt_mv Clustered regularly interspaced short palindromic repeats and CRISPR-associated protein-9 (CRISPR-Cas9) can be used as an efficient tool for genome editing in potato (Solanum tuberosum). From both a scientific and a regulatory perspective, it is beneficial if integration of DNA in the potato genome is avoided. We have implemented a DNA-free genome editing method, using delivery of CRISPR-Cas9 ribonucleoproteins (RNPs) to potato protoplasts, by targeting the gene encoding a granule bound starch synthase (GBSS, EC 2.4.1.242). The RNP method was directly implemented using previously developed protoplast isolation, transfection and regeneration protocols without further adjustments. Cas9 protein was preassembled with RNA produced either synthetically or by in vitro transcription. RNP with synthetically produced RNA (cr-RNP) induced mutations, i.e. indels, at a frequency of up to 9%, with all mutated lines being transgene-free. A mutagenesis frequency of 25% of all regenerated shoots was found when using RNP with in vitro transcriptionally produced RNA (IVT–RNP). However, more than 80% of the shoots with confirmed mutations had unintended inserts in the cut site, which was in the same range as when using DNA delivery. The inserts originated both from DNA template remnants from the in vitro transcription, and from chromosomal potato DNA. In 2–3% of the regenerated shoots from the RNP-experiments, mutations were induced in all four alleles resulting in a complete knockout of the GBSS enzyme function.
Fil: Andersson, Mariette. Swedish University Of Agricultural Sciences; Suecia
Fil: Turesson, Helle. Swedish University Of Agricultural Sciences; Suecia
Fil: Olsson, Niklas. Swedish University Of Agricultural Sciences; Suecia
Fil: Fält, Ann Sofie. Swedish University Of Agricultural Sciences; Suecia
Fil: Ohlsson, Pia. Swedish University Of Agricultural Sciences; Suecia
Fil: Gonzalez, Matías Nicolás. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires Sur. Estación Experimental Agropecuaria Balcarce; Argentina
Fil: Samuelsson, Mathias. Lyckeby Starch AB; Suecia
Fil: Hofvander, Per. Swedish University Of Agricultural Sciences; Suecia
description Clustered regularly interspaced short palindromic repeats and CRISPR-associated protein-9 (CRISPR-Cas9) can be used as an efficient tool for genome editing in potato (Solanum tuberosum). From both a scientific and a regulatory perspective, it is beneficial if integration of DNA in the potato genome is avoided. We have implemented a DNA-free genome editing method, using delivery of CRISPR-Cas9 ribonucleoproteins (RNPs) to potato protoplasts, by targeting the gene encoding a granule bound starch synthase (GBSS, EC 2.4.1.242). The RNP method was directly implemented using previously developed protoplast isolation, transfection and regeneration protocols without further adjustments. Cas9 protein was preassembled with RNA produced either synthetically or by in vitro transcription. RNP with synthetically produced RNA (cr-RNP) induced mutations, i.e. indels, at a frequency of up to 9%, with all mutated lines being transgene-free. A mutagenesis frequency of 25% of all regenerated shoots was found when using RNP with in vitro transcriptionally produced RNA (IVT–RNP). However, more than 80% of the shoots with confirmed mutations had unintended inserts in the cut site, which was in the same range as when using DNA delivery. The inserts originated both from DNA template remnants from the in vitro transcription, and from chromosomal potato DNA. In 2–3% of the regenerated shoots from the RNP-experiments, mutations were induced in all four alleles resulting in a complete knockout of the GBSS enzyme function.
publishDate 2018
dc.date.none.fl_str_mv 2018-12
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/11336/100759
Andersson, Mariette; Turesson, Helle; Olsson, Niklas; Fält, Ann Sofie; Ohlsson, Pia; et al.; Genome editing in potato via CRISPR-Cas9 ribonucleoprotein delivery; Wiley Blackwell Publishing, Inc; Physiologia Plantarum; 164; 4; 12-2018; 378-384
0031-9317
CONICET Digital
CONICET
url http://hdl.handle.net/11336/100759
identifier_str_mv Andersson, Mariette; Turesson, Helle; Olsson, Niklas; Fält, Ann Sofie; Ohlsson, Pia; et al.; Genome editing in potato via CRISPR-Cas9 ribonucleoprotein delivery; Wiley Blackwell Publishing, Inc; Physiologia Plantarum; 164; 4; 12-2018; 378-384
0031-9317
CONICET Digital
CONICET
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/url/http://doi.wiley.com/10.1111/ppl.12731
info:eu-repo/semantics/altIdentifier/doi/10.1111/ppl.12731
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
https://creativecommons.org/licenses/by/2.5/ar/
eu_rights_str_mv openAccess
rights_invalid_str_mv https://creativecommons.org/licenses/by/2.5/ar/
dc.format.none.fl_str_mv application/pdf
application/pdf
dc.publisher.none.fl_str_mv Wiley Blackwell Publishing, Inc
publisher.none.fl_str_mv Wiley Blackwell Publishing, Inc
dc.source.none.fl_str_mv reponame:CONICET Digital (CONICET)
instname:Consejo Nacional de Investigaciones Científicas y Técnicas
reponame_str CONICET Digital (CONICET)
collection CONICET Digital (CONICET)
instname_str Consejo Nacional de Investigaciones Científicas y Técnicas
repository.name.fl_str_mv CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicas
repository.mail.fl_str_mv dasensio@conicet.gov.ar; lcarlino@conicet.gov.ar
_version_ 1842268721587945472
score 13.13397

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