Genome editing in potato with CRISPR/Cas9

Satya Swathi Nadakuduti, Colby G. Starker, Daniel F. Voytas, C. Robin Buell, David S. Douches

Research output: Chapter in Book/Report/Conference proceedingChapter

5 Scopus citations

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.

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages183-201
Number of pages19
DOIs
StatePublished - 2019

Publication series

NameMethods in Molecular Biology
Volume1917
ISSN (Print)1064-3745

Bibliographical note

Funding Information:
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.

Publisher Copyright:
© 2019, Springer Science+Business Media, LLC, part of Springer Nature.

Keywords

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

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