A method for transgene-free genome editing in apple using cell-penetrating peptides
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Date
2025-04
Publication Type
Other Conference Item
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yes
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Abstract
For apple (Malus domestica Borkh.), a wide variety of cultivars exists but only a few meet the high expectations of producers, consumers and retailers. Crossbreeding of apple leads to a reshuffling of cultivar characteristics, rendering the breeding process laborious when introducing favorable traits into a commercially established apple cultivar. Targeted mutagenesis could enhance existing cultivars by disrupting gene function through knockout mutations or by allele replacement. Genome editing with CRISPR/Cas allows for sequence-specific mutation but typically relies on the genomic integration of transgenes encoding the Cas protein and guide RNA. In contrast to integrating transgenes, genome editing components can be delivered into cells as plasmid DNA or as a pre-assembled ribonucleoprotein (RNP), which include Cas protein and guide RNA. Regeneration of plants from the transfected single cells with RNP or plasmid DNA would enable transgene-free genome editing applications. However, there is a trade-off between transfection efficiency and the ability to regenerate apple plants from single cells. Apple cells without a cell wall are readily transfected but fail to regenerate into plantlets, whereas single cells with a cell wall regenerate into apple plantlets but are challenging to transfect. To overcome these limitations, cell-penetrating peptides can be employed to transport cargo molecules into plant cells with a cell wall. We tested the ability of a newly engineered CPP (dTAT-Sar-EED4) to transfect apple single cells containing a cell wall with fluorescent RNP. Single cells treated with the CPP-RNP complex exhibited fluorescent signals inside the cells, unlike cells treated only with fluorescent RNP. Combined with regeneration protocols, CPP-mediated single cell transfection presents a promising method for achieving transgene-free genome editing in apple. This approach has the potential to enhance existing apple cultivars by improving beneficial traits for producers, consumers and retailers.
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published
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Journal / series
Volume
34
Pages / Article No.
34 - 34
Publisher
Schweizer Gesellschaft für Pflanzenbauwissenschaften (SGPW-SSA)
Event
32nd Annual Meeting of the Swiss Society of Agronomy
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Organisational unit
03969 - Studer, Bruno / Studer, Bruno
Notes
Conference presentation hold on April 4, 2025
Funding
176204 - Non-transgenic Genome Editing in Apple (SNF)
Related publications and datasets
Is part of: https://doi.org/10.5281/zenodo.14960342