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Genome Editing in Potato with CRISPR/Cas9

  • Satya Swathi NadakudutiEmail author
  • Colby G. Starker
  • Daniel F. Voytas
  • C. Robin Buell
  • David S. Douches
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1917)

Abstract

Cultivated potato, Solanum tuberosum Group Tuberosum L. (2n = 4x = 48) is a heterozygous tetraploid crop that is clonally propagated, thereby resulting in identical genotypes. Due to the lack of sexual reproduction and its concomitant segregation of alleles, genetic engineering is an efficient way of introducing crop improvement traits in potato. In recent years, genome-editing via the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system for targeted genome modifications has emerged as the most powerful method due to the ease in designing and construction of gene-specific single guide RNA (sgRNA) vectors. These sgRNA vectors are easily reprogrammable to direct Streptococcus pyogenes Cas9 (SpCas9) to generate double stranded breaks (DSBs) in the target genomes that are then repaired by the cell via the error-prone non-homologous end-joining (NHEJ) pathway or by precise homologous recombination (HR) pathway. CRISPR/Cas9 technology has been successfully implemented in potato for targeted mutagenesis to generate knockout mutations (by means of NHEJ) as well as gene targeting to edit an endogenous gene (by HR). In this chapter, we describe procedures for designing sgRNAs, protocols to clone sgRNAs for CRISPR/Cas9 constructs to generate knockouts, design of donor repair templates and use geminivirus replicons (GVRs) to facilitate gene-editing by HR in potato. We also describe tissue culture procedures in potato for Agrobacterium-mediated transformation to generate gene-edited events along with their molecular characterization.

Key words

Potato CRISPR/Cas9 Plant genome-editing Targeted mutagenesis Single guide RNA Agrobacterium-mediated transformation Tissue culture 

Notes

Acknowledgements

This work was supported by the Biotechnology Risk Assessment Grant Program competitive grant no. 2013-33522-21090 from the USDA National Institute of Food and Agriculture (http://nifa.usda.gov/) to David S. Douches, C. Robin Buell and Daniel F. Voytas. Thanks to Felix Encisco and Daniel Zarka for reading the document and their suggestions.

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Satya Swathi Nadakuduti
    • 1
    Email author
  • Colby G. Starker
    • 2
  • Daniel F. Voytas
    • 2
  • C. Robin Buell
    • 3
    • 5
    • 4
  • David S. Douches
    • 1
    • 4
  1. 1.Department of Plant, Soil and Microbial SciencesMichigan State UniversityEast LansingUSA
  2. 2.Department of Genetics, Cell Biology and Development and Center for Genome EngineeringUniversity of MinnesotaMinneapolisUSA
  3. 3.Department of Plant BiologyMichigan State UniversityEast LansingUSA
  4. 4.Michigan State University AgBioResearchMichigan State UniversityEast LansingUSA
  5. 5.Plant Resilience InstituteMichigan State UniversityEast LansingUSA

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