BRIEF RESEARCH REPORT article
Front. Genome Ed.
Sec. Genome Editing in Plants
Volume 7 - 2025 | doi: 10.3389/fgeed.2025.1589431
This article is part of the Research TopicDevelopments in Genetic Transformation and Genome Editing in Horticultural Crops and Their PathogensView all 3 articles
DNA-free CRISPR Genome Editing in Raspberry (Rubus idaeus) Protoplast through RNP-mediated Transfection
Provisionally accepted
- Cranfield University, Cranfield, United Kingdom
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Protoplast-based systems have been utilised in a wide variety of plant species to enable genome editing without chromosomal introgression of foreign DNA into plant genomes.DNA-free genome editing followed by protoplast regeneration allows elite cultivars to be edited without further genetic segregation, preserving their unique genetic composition and their regulatory status as non-transgenic. However, protoplast isolation presents a barrier to the development of advanced breeding technologies in raspberry and no protocol has been published for DNA-free genome editing in the species. Pre-assembled ribonucleoprotein complexes (RNPs) do not require cellular processing and the commercial availability of Cas9 proteins and synthetic guide RNAs has streamlined genome editing protocols. This study presents a novel high-yielding protoplast isolation protocol from raspberry stem cultures and RNP-mediated transfection of protoplast with CRISPR-Cas9. Targeted mutagenesis of the phytoene desaturase gene at two intragenic loci resulted in an editing efficiency of 19%, though estimated efficiency varied depending on the indel analysis technique. Only amplicon sequencing was sensitive enough to confirm genome editing in a low efficiency sample. To our knowledge, this study constitutes the first use of DNA-free genome editing in raspberry protoplast. This protocol provides a valuable platform for understanding gene function and facilitates the future development of precision breeding in this important soft fruit crop.
Keywords: CRISPR-Cas9, Genome editing, Rubus idaeus, DNA-free, RNPs, Protoplasts
Received: 07 Mar 2025; Accepted: 13 Jun 2025.
Copyright: © 2025 Creeth, Thompson and Kevei. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
* Correspondence:
Ryan Creeth, Cranfield University, Cranfield, United Kingdom
Zoltan Kevei, Cranfield University, Cranfield, United Kingdom
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