DNA-free CRISPR gene editing protocol for Solanaceae and Cucurbitaceae crops
Cranfield University offers a DNA-free, protoplast-based CRISPR/Cas9 RNP delivery protocol for gene editing in Solanaceae and Cucurbitaceae crops. This transgene-free approach enables precise genome edits including targeted deletions and inversions in elite cultivars that cannot be edited via traditional Agrobacterium methods due to self-incompatibility or inbreeding depression.
Overview
Many elite crop lines in the Solanaceae and Cucurbitaceae families are difficult to edit using conventional Agrobacterium-mediated CRISPR delivery because they are self-incompatible or suffer from inbreeding depression, making it impossible to eliminate introduced transgenes in subsequent generations. Cranfield University offers a DNA-free gene editing protocol based on ribonucleoprotein (RNP) delivery into plant protoplasts, followed by regeneration of edited cells into whole plants. This approach enables transgene-free genome editing suitable for crops where traditional methods fail, supporting the development of improved cultivars with precise genetic modifications such as targeted deletions and larger chromosomal inversions.
Technical specifications
- DNA-free CRISPR/Cas9 delivery via RNP/PEG transfection into isolated protoplasts, avoiding any introduction of foreign DNA
- Dual gRNA editing capability for generating larger deletions and targeted inversions (anticipated 2Mb inversion in target species)
- Protoplast isolation and editing protocols already validated in raspberry, with successful editing of the PDS gene confirmed in protoplast genomes
- Microcolony production and plant regeneration protocols currently under development, with promising preliminary results
- Target sequence verification by sequencing to confirm gRNA design matches the actual cultivar genome
- Post-regeneration analysis using PCR and sequencing to verify deletion or inversion incidence
- Applicable to multiple species including tomato, raspberry, and other Solanaceae and Cucurbitaceae crops where protoplast regeneration has been demonstrated
Technology readiness level
The protocol builds on several years of gene editing experience at Cranfield University's plant molecular laboratory, beginning with Agrobacterium-mediated editing in tomato and extending to DNA-free RNP delivery in raspberry. Successful protoplast isolation and PDS gene editing in raspberry protoplasts has been completed, with plant regeneration protocols currently in active development. Genome editing via protoplast-based methods has already been demonstrated in several Solanaceae and Cucurbitaceae species, providing a strong foundation. The technology readiness level is approximately TRL 4-5, with core editing steps validated in laboratory settings and plant regeneration under optimization. The research team has access to all necessary laboratory facilities at Cranfield University to carry out the full workflow.
About Cranfield University
Cranfield University is a postgraduate‑only, research‑intensive public institution in Bedfordshire, known for applied, industry‑engaged research and professional education. Assets include a co‑located airport, full‑scale laboratories and pilot plants, and a research and technology park that places companies alongside researchers. A defense and security campus, deep partnerships with global firms, and extensive executive education create direct routes for collaboration, sponsored studentships, and workforce upskilling. Research is supported by UK Research and Innovation councils and Innovate UK, with additional backing from defense stakeholders and competitive European programs. A dedicated technology transfer office manages IP, licensing, spin‑outs, and incubation space within the research park.
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