Abstract
Meganucleases are rare cutting enzymes that can generate DNA modifications and are part of the plant genome editing toolkit although they lack versatility. Here, we evaluated the use of two meganucleases, I-SceI and a customized meganuclease, in tomato and oilseed rape. Different strategies were explored for the use of these meganucleases. The activity of a customized and a I-SceI meganucleases was first estimated by the use of a reporter construct GFFP with the target sequences and enabled to demonstrate that both meganucleases can generate double-strand break and HDR mediated recombination in a reporter gene. Interestingly, I-SceI seems to have a higher DSB efficiency than the customized meganuclease: up to 62.5% in tomato and 44.8% in oilseed rape. Secondly, the same exogenous landing pad was introduced in both species. Despite being less efficient compared to I-SceI, the customized meganuclease was able to generate the excision of an exogenous transgene (large deletion of up to 3316 bp) present in tomato. In this paper, we also present some pitfalls to be considered before using meganucleases (e.g., potential toxicity) for plant genome editing.
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Acknowledgements
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 authors thank Peter Rogowsky and Paul Wyatt for their efficient management of the GENIUS project, for providing vectors with CMN and I-SceI respectively and for their contribution and review to the present study. The authors thank Cellectis for providing the meganuclease sequences and their expertise in the genome editing field. The authors thanks Emmanuel Botton for his work with tomato plants.
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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).
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Danilo, B., Montes, É., Archambeau, H. et al. I-SceI and customized meganucleases-mediated genome editing in tomato and oilseed rape. Transgenic Res 31, 87–105 (2022). https://doi.org/10.1007/s11248-021-00287-2
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DOI: https://doi.org/10.1007/s11248-021-00287-2