Efficient CRISPR/Cas9 genome editing in alfalfa using a public germplasm
Because its ability to acquire large amounts of nitrogen by symbiosis, tetraploid alfalfa is the main source of vegetable proteins in meat and milk production systems in temperate regions. Alfalfa cultivation also adds fixed nitrogen to the soil, improving the production of non-legumes in crop rotat...
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| Format: | info:ar-repo/semantics/artículo |
| Language: | Inglés |
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Frontiers Media
2024
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| Online Access: | http://hdl.handle.net/20.500.12123/16599 https://www.frontiersin.org/articles/10.3389/fagro.2021.661526/full https://doi.org/10.3389/fagro.2021.661526 |
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| author | Bottero, Ana Emilia Massa, Gabriela Alejandra Gonzalez, Matías Nicolás Stritzler, Margarita Tajima, Hiromi Gomez, Maria Cristina Frare, Romina Alejandra Feingold, Sergio Enrique Blumwald, Eduardo Ayub, Nicolás Daniel Soto, Gabriela Cynthia |
| author_browse | Ayub, Nicolás Daniel Blumwald, Eduardo Bottero, Ana Emilia Feingold, Sergio Enrique Frare, Romina Alejandra Gomez, Maria Cristina Gonzalez, Matías Nicolás Massa, Gabriela Alejandra Soto, Gabriela Cynthia Stritzler, Margarita Tajima, Hiromi |
| author_facet | Bottero, Ana Emilia Massa, Gabriela Alejandra Gonzalez, Matías Nicolás Stritzler, Margarita Tajima, Hiromi Gomez, Maria Cristina Frare, Romina Alejandra Feingold, Sergio Enrique Blumwald, Eduardo Ayub, Nicolás Daniel Soto, Gabriela Cynthia |
| author_sort | Bottero, Ana Emilia |
| collection | INTA Digital |
| description | Because its ability to acquire large amounts of nitrogen by symbiosis, tetraploid alfalfa is the main source of vegetable proteins in meat and milk production systems in temperate regions. Alfalfa cultivation also adds fixed nitrogen to the soil, improving the production of non-legumes in crop rotation and reducing the use of nitrogen fertilizers derived from fossil fuel. Despite its economic and ecological relevance, alfalfa genetics remains poorly understood, limiting the development of public elite germplasm. In this brief article, we reported the high-efficiency of alfalfa mutagenesis by using the public clone C23 and the CRISPR/Cas9 system. Around half of the GUS overexpressing plants (35S-GUS under C23 genomic background) transformed with an editing plasmid containing two sgRNAs against the GUS gene and the Cas9 nuclease exhibited absence of GUS activity. Nucleotide analysis showed that the inactivation of GUS in CRISPR/Cas9-editing events were produced via different modifications in the GUS gene, including frameshift and non-sense mutations. Using the CRISPR/Cas9 system and two sgRNAs, we have also edited the alfalfa gene NOD26, generating plants with different doses of alleles at this locus, including complete gene knockout at high efficiency (11%). Finally, we discuss the potential applications of genome-editing technologies to polyploid research and to alfalfa improvement public programs. |
| format | info:ar-repo/semantics/artículo |
| id | INTA16599 |
| institution | Instituto Nacional de Tecnología Agropecuaria (INTA -Argentina) |
| language | Inglés |
| publishDate | 2024 |
| publishDateRange | 2024 |
| publishDateSort | 2024 |
| publisher | Frontiers Media |
| publisherStr | Frontiers Media |
| record_format | dspace |
| spelling | INTA165992024-02-14T15:02:50Z Efficient CRISPR/Cas9 genome editing in alfalfa using a public germplasm Bottero, Ana Emilia Massa, Gabriela Alejandra Gonzalez, Matías Nicolás Stritzler, Margarita Tajima, Hiromi Gomez, Maria Cristina Frare, Romina Alejandra Feingold, Sergio Enrique Blumwald, Eduardo Ayub, Nicolás Daniel Soto, Gabriela Cynthia Medicago sativa Edición de Genes Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Interespaciadas Mutantes Germoplasma Gene Editing CRISPR Mutants Germplasm Alfalfa Lucerne Because its ability to acquire large amounts of nitrogen by symbiosis, tetraploid alfalfa is the main source of vegetable proteins in meat and milk production systems in temperate regions. Alfalfa cultivation also adds fixed nitrogen to the soil, improving the production of non-legumes in crop rotation and reducing the use of nitrogen fertilizers derived from fossil fuel. Despite its economic and ecological relevance, alfalfa genetics remains poorly understood, limiting the development of public elite germplasm. In this brief article, we reported the high-efficiency of alfalfa mutagenesis by using the public clone C23 and the CRISPR/Cas9 system. Around half of the GUS overexpressing plants (35S-GUS under C23 genomic background) transformed with an editing plasmid containing two sgRNAs against the GUS gene and the Cas9 nuclease exhibited absence of GUS activity. Nucleotide analysis showed that the inactivation of GUS in CRISPR/Cas9-editing events were produced via different modifications in the GUS gene, including frameshift and non-sense mutations. Using the CRISPR/Cas9 system and two sgRNAs, we have also edited the alfalfa gene NOD26, generating plants with different doses of alleles at this locus, including complete gene knockout at high efficiency (11%). Finally, we discuss the potential applications of genome-editing technologies to polyploid research and to alfalfa improvement public programs. Instituto de Biotecnología Fil: Bottero, Ana Emilia. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; Argentina Fil: Bottero, Ana Emilia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Bottero, Ana Emilia. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Genética; Argentina Fil: Massa, Gabriela Alejandra. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce. Instituto de Innovación para la Producción Agropecuaria y el Desarrollo Sostenible (IPADS); Argentina Fil: Massa, Gabriela Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Massa, Gabriela Alejandra. Universidad Nacional de Mar del Plata. Facultad de Ciencias Agrarias; Argentina Fil: González, Matías Nicolás. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce. Instituto de Innovación para la Producción Agropecuaria y el Desarrollo Sostenible (IPADS); Argentina Fil: González, Matías Nicolás. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Stritzler, Margarita. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; Argentina Fil: Stritzler, Margarita. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Stritzler, Margarita. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Genética; Argentina Fil: Tajima, Hiromi. University of California, Davis. Department of Plant Sciences; Estados Unidos Fil: Gomez, Maria Cristina. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; Argentina Fil: Gomez, Maria Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Gomez, Maria Cristina. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Genética; Argentina Fil: Frare, Romina Alejandra. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; Argentina Fil: Frare, Romina Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Frare, Romina Alejandra. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Genética; Argentina Fil: Feingold, Sergio Enrique. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce. Instituto de Innovación para la Producción Agropecuaria y el Desarrollo Sostenible (IPADS); Argentina Fil: Feingold, Sergio Enrique. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Blumwald, Eduardo. University of California, Davis. Department of Plant Sciences; Estados Unidos Fil: Ayub, Nicolás Daniel. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; Argentina Fil: Ayub, Nicolás Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Ayub, Nicolás Daniel. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Genética; Argentina Fil: Soto, Gabriela Cynthia. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; Argentina Fil: Soto, Gabriela Cynthia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Soto, Gabriela Cynthia. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Genética; Argentina 2024-02-14T14:45:15Z 2024-02-14T14:45:15Z 2021-07 info:ar-repo/semantics/artículo info:eu-repo/semantics/article info:eu-repo/semantics/publishedVersion http://hdl.handle.net/20.500.12123/16599 https://www.frontiersin.org/articles/10.3389/fagro.2021.661526/full 2673-3218 https://doi.org/10.3389/fagro.2021.661526 eng info:eu-repograntAgreement/INTA/2019-PE-E6-I115-001, Edición génica, transgénesis y mutagénesis como generadores de nueva variabilidad en especies de interés agropecuario Frontiers in Agronomy 3 : 661526 (Julio 2021) info:eu-repo/semantics/openAccess 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 Frontiers Media |
| spellingShingle | Medicago sativa Edición de Genes Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Interespaciadas Mutantes Germoplasma Gene Editing CRISPR Mutants Germplasm Alfalfa Lucerne Bottero, Ana Emilia Massa, Gabriela Alejandra Gonzalez, Matías Nicolás Stritzler, Margarita Tajima, Hiromi Gomez, Maria Cristina Frare, Romina Alejandra Feingold, Sergio Enrique Blumwald, Eduardo Ayub, Nicolás Daniel Soto, Gabriela Cynthia Efficient CRISPR/Cas9 genome editing in alfalfa using a public germplasm |
| title | Efficient CRISPR/Cas9 genome editing in alfalfa using a public germplasm |
| title_full | Efficient CRISPR/Cas9 genome editing in alfalfa using a public germplasm |
| title_fullStr | Efficient CRISPR/Cas9 genome editing in alfalfa using a public germplasm |
| title_full_unstemmed | Efficient CRISPR/Cas9 genome editing in alfalfa using a public germplasm |
| title_short | Efficient CRISPR/Cas9 genome editing in alfalfa using a public germplasm |
| title_sort | efficient crispr cas9 genome editing in alfalfa using a public germplasm |
| topic | Medicago sativa Edición de Genes Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Interespaciadas Mutantes Germoplasma Gene Editing CRISPR Mutants Germplasm Alfalfa Lucerne |
| url | http://hdl.handle.net/20.500.12123/16599 https://www.frontiersin.org/articles/10.3389/fagro.2021.661526/full https://doi.org/10.3389/fagro.2021.661526 |
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