The DFR locus: A smart landing pad for targeted transgene insertion in tomato

PLoS One. 2018 Dec 6;13(12):e0208395. doi: 10.1371/journal.pone.0208395. eCollection 2018.

Abstract

Targeted insertion of transgenes in plants is still challenging and requires further technical innovation. In the present study, we chose the tomato DFR gene involved in anthocyanin biosynthesis as a landing pad for targeted transgene insertion using CRISPR-Cas9 in a two-step strategy. First, a 1013 bp was deleted in the endogenous DFR gene. Hypocotyls and callus of in vitro regenerated plantlets homozygous for the deletion were green instead of the usual anthocyanin produced purple colour. Next, standard Agrobacterium-mediated transformation was used to target transgene insertion at the DFR landing pad in the dfr deletion line. The single binary vector carried two sgRNAs, a donor template containing two homology arms of 400 bp, the previously deleted DFR sequence, and a NptII expression cassette. Regenerating plantlets were screened for a purple-colour phenotype indicating that DFR function had been restored. Targeted insertions were identified in 1.29% of the transformed explants. Thus, we established an efficient method for selecting HDR-mediated transgene insertion using the CRISPR-Cas9 system in tomato. The visual screen used here facilitates selection of these rare gene targeting events, does not necessitate the systematic PCR screening of all the regenerating material and can be potentially applied to other crops.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Agrobacterium / genetics
  • Anthocyanins / biosynthesis
  • Gene Editing*
  • Plant Proteins / genetics*
  • Plants, Genetically Modified / anatomy & histology
  • Plants, Genetically Modified / growth & development
  • Plants, Genetically Modified / microbiology
  • RNA, Guide, CRISPR-Cas Systems / genetics
  • Sequence Deletion
  • Solanum lycopersicum / anatomy & histology*
  • Solanum lycopersicum / genetics*
  • Solanum lycopersicum / growth & development
  • Solanum lycopersicum / microbiology
  • Transformation, Bacterial
  • Transgenes

Substances

  • Anthocyanins
  • Plant Proteins
  • RNA, Guide, CRISPR-Cas Systems

Grants and funding

This work was funded by the Investissement d’Avenir program of the French National Agency of Research for the project GENIUS (ANR-11-BTBR-0001_GENIUS). The IJPB benefits from the support of the LabEx Saclay Plant Sciences-SPS (ANR-10-LABX-0040-SPS). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.