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Capítulo de Libro

CRISPR/Cas9 for potato functional genomics and breeding

Título del libro: Plant Genome engineering

Gonzalez, Matías NicolásIcon ; Massa, Gabriela AlejandraIcon ; Andersson, Mariette; Storani, LeonardoIcon ; Olsson, Niklas; Décima Oneto, Cecilia Andrea; Hofvander, Per; Feingold, Sergio
Otros responsables: Yang, Bing; Harwood, Wendy; Que, Qiudeng
Fecha de publicación: 2023
Editorial: Humana Press
ISBN: 978-1-0716-3130-0
Idioma: Inglés
Clasificación temática:
Biotecnología Agrícola y Biotecnología Alimentaria

Resumen

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.
Palabras clave: POTATO , CRISPR/CAS9 , GENOME EDITING , FUNCTIONAL GENOMICS , CROP BREEDING , AGROBACTERIUM TUMEFACIENS , PROTOPLASTS , RIBONUCLEOPROTEINS
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info:eu-repo/semantics/restrictedAccess Excepto donde se diga explícitamente, este item se publica bajo la siguiente descripción: Creative Commons Attribution-NonCommercial-ShareAlike 2.5 Unported (CC BY-NC-SA 2.5)
Identificadores
URI: http://hdl.handle.net/11336/234818
URL: https://link.springer.com/protocol/10.1007/978-1-0716-3131-7_21
Colecciones
Capítulos de libros (IPADS BALCARCE)
Capítulos de libros de INSTITUTO DE INNOVACIÓN PARA LA PRODUCCIÓN AGROPECUARIA Y EL DESARROLLO SOSTENIBLE
Citación
Gonzalez, Matías Nicolás; Massa, Gabriela Alejandra; Andersson, Mariette; Storani, Leonardo; Olsson, Niklas; et al.; CRISPR/Cas9 for potato functional genomics and breeding; Humana Press; 2653; 2023; 333-363
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