DNA-free CRISPR editing for climate-resilient grapevines with delayed ripening
Genome editing platform using Cas9-sgRNA RNP delivery into grapevine protoplasts to produce transgene-free edited plants of cv Garganega with postponed berry ripening, addressing climate change impacts on vineyards.
Overview
Climate change is causing early berry ripening in vineyards, producing grapes with unbalanced composition at harvest. This solution applies DNA-free CRISPR/Cas9 genome editing to grapevine protoplasts to generate transgene-free edited plants of the local Italian variety Garganega, targeting regulatory regions of candidate ripening regulators. The goal is to postpone the onset of ripening, enabling vineyards to adapt to warming conditions while preserving varietal identity. Plants are regenerated from single cells, avoiding chimeras and producing refined cis-regulatory alleles that can fine-tune the duration of the vegetative phase.
Technical specifications
- DNA-free editing approach: Preassembled Cas9-sgRNA ribonucleoprotein (RNP) complexes are delivered directly into protoplasts isolated from embryogenic callus, leaving no foreign DNA in the final plant
- Targeted regulatory regions: Guide RNAs are designed to target cis-acting elements in regulatory regions of candidate ripening regulators, generating diverse cis-regulatory alleles rather than knockout mutations
- Single-cell regeneration: Whole plants are regenerated from single edited protoplasts, avoiding chimeras and ensuring uniform edits
- Proven regeneration pipeline: Protoplast isolation from embryogenic callus followed by regeneration into whole Garganega plants with wild-type morphology and growth habit
- Functional validation precedent: Stable overexpression of candidate regulators confirmed increased chlorophyll degradation, anthocyanin accumulation, and induction of ripening-related genes
- Proof-of-concept demonstrated: GFP reporter gene knockout via RNP delivery confirmed CRISPR/Cas9 activity in grapevine protoplasts
- Timeline: Whole plant regeneration achievable in approximately 12 months
Technology readiness level
The technology is at an advanced validation stage. The lab has demonstrated functional characterization of candidate ripening regulators through transgenic overexpression studies and has successfully applied DNA-free RNP-based editing in grapevine protoplasts with confirmed regeneration of whole plants. A proof-of-concept GFP knockout validated the editing workflow. The next phase involves targeting endogenous ripening-regulator loci in cv Garganega protoplasts to produce edited transgene-free plants with delayed ripening, followed by field validation of the regenerated lines.
About University of Verona
The University of Verona is a comprehensive public research university in northern Italy, combining broad academic breadth with a strong clinical presence through its integrated university hospital. Industry collaborates through contract research and sponsored testing supported by on-campus core facilities and co-located clinical environments that enable rapid translation and validation. Companies benefit from proximity to the Veneto region’s dense manufacturing and logistics ecosystem, allowing agile pilots and joint development, while structured internships and thesis placements create a reliable talent pipeline. Research is supported by competitive European and national funding, including Horizon Europe and programs from Italy’s ministries and regional authorities. A dedicated technology transfer office manages IP, licensing, sponsored research, and startup support with streamlined engagement for corporate partners.
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