Abstract
The possibility to produce plants edited in multiple genes by means of DNA-free approaches opens new perspectives for breeding purposes and acceptance of resultant genotypes. In this work, we have explored the polyethylene glycol (PEG)-mediated delivery of ribonucleoproteins (RNPs) in tomato protoplasts using a multiplexing approach (i.e. two genes targeted simultaneously using two sgRNAs per gene) for the first time. We have analysed the editing outcome in fully developed green calli and demonstrated that tomato protoplasts are a valid cell target for RNP-mediated multiplexed genome editing with high efficiency.
Key message
RNP could be applied with high efficiency in a multiplexing genome editing approach in tomato protoplasts.
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Abbreviations
- RNP:
-
Ribonucleoprotein
- HFRA:
-
High resolution fragment analysis
- PEG:
-
Polyethylene glycol
- CRISPR:
-
Clustered regularly interspaced short palindromic repeats
- Cas9:
-
CRISPR associated protein 9
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Acknowledgements
The authors wish to acknowledge Mirela Beganovic and Ann-Sofie Fält for their help with in vitro culture. The authors would also like to thank Sjur Sandgrind for his help with the confocal microscope. The authors acknowledge Nunzio D’Agostino for the design of the sgRNAs and Francesco Camerlengo for the tomato hairy roots data.
Funding
The work presented here was financially supported by the Italian Ministry of Agriculture (MiPAAF), project BIOTECH-Cisget, and Formas, a Swedish government research council for sustainable development (2018-01459). Alessandro Nicolia acknowledges the financial support received from Italian Council for Agricultural Research and Economics (CREA) for the short mobility grant during his visit at the Swedish University of Agricultural Sciences (SLU)—Alnarp.
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AN, MA, PH and TC conceived the experiment and contributed to manuscript preparation. AN, MA and GF carried out experimental activities.
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Communicated by Joyce Van Eck.
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Nicolia, A., Andersson, M., Hofvander, P. et al. Tomato protoplasts as cell target for ribonucleoprotein (RNP)-mediated multiplexed genome editing. Plant Cell Tiss Organ Cult 144, 463–467 (2021). https://doi.org/10.1007/s11240-020-01954-8
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DOI: https://doi.org/10.1007/s11240-020-01954-8