CRISPR/Cas9 for potato functional genomics and breeding
Cultivated potato (Solanum tuberosum L.) is one of the most important staple food crops worldwide. Its tetraploid and highly heterozygous nature pose a great challenge to its basic research and trait improvement through traditional mutagenesis and/or crossbreeding. The establishment of the clustered...
| Main Authors: | , , , , , , , |
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| Format: | Capítulo de libro |
| Language: | Inglés |
| Published: |
Humana Press (Springer)
2024
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| Subjects: | |
| Online Access: | http://hdl.handle.net/20.500.12123/17657 https://link.springer.com/protocol/10.1007/978-1-0716-3131-7_21 https://doi.org/10.1007/978-1-0716-3131-7_21 |
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| author | Gonzalez, Matías Nicolás Massa, Gabriela Alejandra Andersson, Mariette Storani, Leonardo Olsson, Niklas Decima Oneto, Cecilia Andrea Hofvander, Per Feingold, Sergio Enrique |
| author_browse | Andersson, Mariette Decima Oneto, Cecilia Andrea Feingold, Sergio Enrique Gonzalez, Matías Nicolás Hofvander, Per Massa, Gabriela Alejandra Olsson, Niklas Storani, Leonardo |
| author_facet | Gonzalez, Matías Nicolás Massa, Gabriela Alejandra Andersson, Mariette Storani, Leonardo Olsson, Niklas Decima Oneto, Cecilia Andrea Hofvander, Per Feingold, Sergio Enrique |
| author_sort | Gonzalez, Matías Nicolás |
| collection | INTA Digital |
| description | Cultivated potato (Solanum tuberosum L.) is one of the most important staple food crops worldwide. Its tetraploid and highly heterozygous nature pose a great challenge to its basic research and trait improvement through traditional mutagenesis and/or crossbreeding. The establishment of the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) as a gene-editing tool has allowed the alteration of specific gene sequences and their concomitant gene function, providing powerful technology for potato gene functional analysis and improvement of elite cultivars. This technology relies on a short RNA molecule called single guide RNA (sgRNA) that directs the Cas9 nuclease to induce a site-specific double-stranded break (DSB). Further, repair of the DSB by the error-prone non-homologous end joining (NHEJ) mechanism, leads to the introduction of targeted mutations, which can be used to produce the loss of function of specific gene/s. In this chapter, we describe experimental procedures to apply the CRISPR/Cas9 technology for potato genome editing. First, we provide strategies for target selection and sgRNA design and describe a Golden Gate-based cloning system to obtain a sgRNA/Cas9-encoding binary vector. We also describe an optimized protocol for ribonucleoprotein complexes (RNP) assembly. The binary vector can be used for both Agrobacterium-mediated transformation and transient expression in potato protoplasts, while the RNP complexes are intended to obtain edited potato lines through protoplast transfection and plant regeneration. Finally, we describe procedures to identify the gene-edited potato lines. The methods described here are suitable for potato gene functional analysis and breeding. |
| format | Capítulo de libro |
| id | INTA17657 |
| institution | Instituto Nacional de Tecnología Agropecuaria (INTA -Argentina) |
| language | Inglés |
| publishDate | 2024 |
| publishDateRange | 2024 |
| publishDateSort | 2024 |
| publisher | Humana Press (Springer) |
| publisherStr | Humana Press (Springer) |
| record_format | dspace |
| spelling | INTA176572024-05-08T11:28:39Z CRISPR/Cas9 for potato functional genomics and breeding Gonzalez, Matías Nicolás Massa, Gabriela Alejandra Andersson, Mariette Storani, Leonardo Olsson, Niklas Decima Oneto, Cecilia Andrea Hofvander, Per Feingold, Sergio Enrique Papa Edición de Genes Genómica Funcional Agrobacterium tumefaciens Proteínas Mejoramiento Genético Potatoes Gene Editing Functional Genomics Proteins Genetic Improvement CRISPR Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Interespaciadas Cultivated potato (Solanum tuberosum L.) is one of the most important staple food crops worldwide. Its tetraploid and highly heterozygous nature pose a great challenge to its basic research and trait improvement through traditional mutagenesis and/or crossbreeding. The establishment of the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) as a gene-editing tool has allowed the alteration of specific gene sequences and their concomitant gene function, providing powerful technology for potato gene functional analysis and improvement of elite cultivars. This technology relies on a short RNA molecule called single guide RNA (sgRNA) that directs the Cas9 nuclease to induce a site-specific double-stranded break (DSB). Further, repair of the DSB by the error-prone non-homologous end joining (NHEJ) mechanism, leads to the introduction of targeted mutations, which can be used to produce the loss of function of specific gene/s. In this chapter, we describe experimental procedures to apply the CRISPR/Cas9 technology for potato genome editing. First, we provide strategies for target selection and sgRNA design and describe a Golden Gate-based cloning system to obtain a sgRNA/Cas9-encoding binary vector. We also describe an optimized protocol for ribonucleoprotein complexes (RNP) assembly. The binary vector can be used for both Agrobacterium-mediated transformation and transient expression in potato protoplasts, while the RNP complexes are intended to obtain edited potato lines through protoplast transfection and plant regeneration. Finally, we describe procedures to identify the gene-edited potato lines. The methods described here are suitable for potato gene functional analysis and breeding. 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 Balcarce; 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 Balcarce; Argentina Fil: Massa, Gabriela Alejandra. Universidad Nacional de Mar del Plata. Facultad de Ciencias Agrarias; Argentina Fil: Andersson, Mariette. Swedish University Of Agricultural Sciences; 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 Balcarce; Argentina Fil: Olsson, Niklas. Swedish University Of Agricultural Sciences; 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 Balcarce; Argentina Fil: Décima Oneto, Cecilia Andrea.Universidad Nacional de Mar del Plata. Facultad de Ciencias Agrarias; Argentina Fil: Hofvander, Per. Swedish University Of Agricultural Sciences; Suecia Fil: Feingold, Sergio. 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 Balcarce; Argentina 2024-05-08T11:14:58Z 2024-05-08T11:14:58Z 2023-03-31 info:ar-repo/semantics/parte de libro info:eu-repo/semantics/bookPart info:eu-repo/semantics/publishedVersion http://hdl.handle.net/20.500.12123/17657 https://link.springer.com/protocol/10.1007/978-1-0716-3131-7_21 978-1-0716-3130-0 978-1-0716-3131-7 https://doi.org/10.1007/978-1-0716-3131-7_21 eng info:eu-repo/semantics/restrictedAccess http://creativecommons.org/licenses/by-nc-sa/4.0/ Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International (CC BY-NC-SA 4.0) application/pdf Humana Press (Springer) Plant Genome engineering / Yang, B.; Harwood, W.; Que, Q. (editors). New York: Humana Press, 2023. Chapter 21, p. 333-361 |
| spellingShingle | Papa Edición de Genes Genómica Funcional Agrobacterium tumefaciens Proteínas Mejoramiento Genético Potatoes Gene Editing Functional Genomics Proteins Genetic Improvement CRISPR Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Interespaciadas Gonzalez, Matías Nicolás Massa, Gabriela Alejandra Andersson, Mariette Storani, Leonardo Olsson, Niklas Decima Oneto, Cecilia Andrea Hofvander, Per Feingold, Sergio Enrique CRISPR/Cas9 for potato functional genomics and breeding |
| title | CRISPR/Cas9 for potato functional genomics and breeding |
| title_full | CRISPR/Cas9 for potato functional genomics and breeding |
| title_fullStr | CRISPR/Cas9 for potato functional genomics and breeding |
| title_full_unstemmed | CRISPR/Cas9 for potato functional genomics and breeding |
| title_short | CRISPR/Cas9 for potato functional genomics and breeding |
| title_sort | crispr cas9 for potato functional genomics and breeding |
| topic | Papa Edición de Genes Genómica Funcional Agrobacterium tumefaciens Proteínas Mejoramiento Genético Potatoes Gene Editing Functional Genomics Proteins Genetic Improvement CRISPR Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Interespaciadas |
| url | http://hdl.handle.net/20.500.12123/17657 https://link.springer.com/protocol/10.1007/978-1-0716-3131-7_21 https://doi.org/10.1007/978-1-0716-3131-7_21 |
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