The DFR locus: A smart landing pad for targeted transgene insertion in tomato.
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...
| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Public Library of Science (PLoS)
2018-01-01
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| Series: | PLoS ONE |
| Online Access: | https://doi.org/10.1371/journal.pone.0208395 |
| _version_ | 1818400217284739072 |
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| author | Benoit Danilo Laura Perrot Emmanuel Botton Fabien Nogué Marianne Mazier |
| author_facet | Benoit Danilo Laura Perrot Emmanuel Botton Fabien Nogué Marianne Mazier |
| author_sort | Benoit Danilo |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-12-14T07:33:04Z |
| format | Article |
| id | doaj.art-0da0f52fdd7a439da8bde661a5b8bc54 |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-12-14T07:33:04Z |
| publishDate | 2018-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-0da0f52fdd7a439da8bde661a5b8bc542022-12-21T23:11:19ZengPublic Library of Science (PLoS)PLoS ONE1932-62032018-01-011312e020839510.1371/journal.pone.0208395The DFR locus: A smart landing pad for targeted transgene insertion in tomato.Benoit DaniloLaura PerrotEmmanuel BottonFabien NoguéMarianne MazierTargeted 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.https://doi.org/10.1371/journal.pone.0208395 |
| spellingShingle | Benoit Danilo Laura Perrot Emmanuel Botton Fabien Nogué Marianne Mazier The DFR locus: A smart landing pad for targeted transgene insertion in tomato. PLoS ONE |
| title | The DFR locus: A smart landing pad for targeted transgene insertion in tomato. |
| title_full | The DFR locus: A smart landing pad for targeted transgene insertion in tomato. |
| title_fullStr | The DFR locus: A smart landing pad for targeted transgene insertion in tomato. |
| title_full_unstemmed | The DFR locus: A smart landing pad for targeted transgene insertion in tomato. |
| title_short | The DFR locus: A smart landing pad for targeted transgene insertion in tomato. |
| title_sort | dfr locus a smart landing pad for targeted transgene insertion in tomato |
| url | https://doi.org/10.1371/journal.pone.0208395 |
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