Geminivirus-mediated genome editing in potato (Solanum tuberosum l.) using sequence-specific nucleases

Nathaniel M. Butler, Nicholas J. Baltes, Daniel F. Voytas, David S. Douches

Research output: Contribution to journalArticlepeer-review

96 Scopus citations

Abstract

Genome editing using sequence-specific nucleases (SSNs) is rapidly being developed for genetic engineering in crop species. The utilization of zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats/CRISPR-associated systems (CRISPR/Cas) for inducing double-strand breaks facilitates targeting of virtually any sequence for modification. Targeted mutagenesis via non-homologous end-joining (NHEJ) has been demonstrated extensively as being the preferred DNA repair pathway in plants. However, gene targeting via homologous recombination (HR) remains more elusive but could be a powerful tool for directed DNA repair. To overcome barriers associated with gene targeting, a geminivirus replicon (GVR) was used to deliver SSNs targeting the potato ACETOLACTATE SYNTHASE1 (ALS1) gene and repair templates designed to incorporate herbicide-inhibiting point mutations within the ALS1 locus. Transformed events modified with GVRs held point mutations that were capable of supporting a reduced herbicide susceptibility phenotype, while events transformed with conventional T-DNAs held no detectable mutations and were similar to wild-type. Regeneration of transformed events improved detection of point mutations that supported a stronger reduced herbicide susceptibility phenotype. These results demonstrate the use of geminiviruses for delivering genome editing reagents in plant species, and a novel approach to gene targeting in a vegetatively propagated species.

Original languageEnglish (US)
Article number1045
JournalFrontiers in Plant Science
Volume7
Issue numberJULY2016
DOIs
StatePublished - Jul 21 2016

Keywords

  • Acetolactate synthase
  • Bean yellow dwarf virus
  • CRISPR/Cas
  • Gene replacement
  • Gene targeting
  • Homologous recombination
  • TALEN
  • ZFN

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