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CRISPR/Cas9 Technology for Potato Functional Genomics and Breeding

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Plant Genome Engineering

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2653))

Abstract

Cultivated potato (Solanum tuberosum L.) is one of the most important staple food crops worldwide. Its tetraploid and highly heterozygous nature poses 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 (RNP) complex 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.

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Author information

Author notes

  1. Per Hofvander and Sergio Enrique Feingold are joint senior authors.

Authors and Affiliations

  1. Laboratorio de Agrobiotecnología, IPADS (INTA – CONICET), Balcarce, Argentina

    Matías Nicolás González, Gabriela Alejandra Massa, Leonardo Storani, Cecilia Andrea Décima Oneto & Sergio Enrique Feingold

  2. Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Buenos Aires, Argentina

    Matías Nicolás González & Gabriela Alejandra Massa

  3. Facultad de Ciencias Agrarias, Universidad Nacional de Mar del Plata, Balcarce, Argentina

    Gabriela Alejandra Massa & Cecilia Andrea Décima Oneto

  4. Department of Plant Breeding, Swedish University of Agricultural Sciences, Lomma, Sweden

    Mariette Andersson, Niklas Olsson & Per Hofvander

  5. Agencia Nacional de Promoción Científica y Tecnológica, Buenos Aires, Argentina

    Leonardo Storani

Authors
  1. Matías Nicolás González
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  2. Gabriela Alejandra Massa
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  3. Mariette Andersson
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  4. Leonardo Storani
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  5. Niklas Olsson
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  6. Cecilia Andrea Décima Oneto
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  7. Per Hofvander
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  8. Sergio Enrique Feingold
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Corresponding author

Correspondence to Matías Nicolás González .

Editor information

Editors and Affiliations

  1. Division of Plant Sciences, University of Missouri, Columbia, MO, USA

    Bing Yang

  2. Department of Crop Genetics, John Innes Centre, Norwich, UK

    Wendy Harwood

  3. Syngenta Crop Protection, LLC., Research Triangle Park, NC, USA

    Qiudeng Que

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© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

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González, M.N. et al. (2023). CRISPR/Cas9 Technology for Potato Functional Genomics and Breeding. In: Yang, B., Harwood, W., Que, Q. (eds) Plant Genome Engineering. Methods in Molecular Biology, vol 2653. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3131-7_21

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  • DOI: https://doi.org/10.1007/978-1-0716-3131-7_21

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-3130-0

  • Online ISBN: 978-1-0716-3131-7

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