Ribonucleoprotein-mediated genome editing in crop protoplasts for chromosomal inversion
A DNA-free genome editing approach using pre-assembled guide RNA/Cas9 ribonucleoprotein complexes co-delivered into plant protoplasts to induce targeted chromosomal inversions. Fluorescence-activated cell sorting enables high-throughput screening of successfully edited protoplasts for regeneration into edited plants.
Overview
This solution offers a rapid, DNA-free genome editing method for crop plants using ribonucleoprotein (RNP) complexes delivered directly into plant protoplasts. By co-transfecting two pre-assembled guide RNA/Cas9 complexes, the approach creates targeted double-strand breaks that induce precise chromosomal inversions. Fluorescence-activated cell sorting (FACS) is used to isolate successfully co-transfected protoplasts, which are then regenerated into whole plants. The method is particularly valuable for crops lacking standardised transformation and regeneration protocols, including species in the Solanaceae and Cucurbitaceae families. Compared to conventional plasmid-driven approaches, this technique reduces off-target editing risks, eliminates cloning steps, and accelerates timelines from protoplast isolation to confirmed edited plants.
Technical specifications
Key features:
- DNA-free editing using pre-assembled ribonucleoprotein complexes composed of guide RNA and fluorescently labelled Cas9
- Co-delivery of two distinct RNP complexes to create simultaneous double-strand breaks for chromosomal inversion
- Protoplast isolation from young leaf tissue, independent of crop life cycle constraints
- Fluorescence-based high-throughput screening and sorting (FACS) to identify and enrich co-transfected protoplasts
- Regeneration pipeline converting sorted protoplasts into seedlings and mature plants
- DNA sequencing-based confirmation of intended edits and screening for off-target effects
- Applicable to Solanaceae and Cucurbitaceae crops where established protoplast isolation, transfection, and regeneration protocols exist
Infrastructure and expertise available:
- Dedicated genome editing and microscopy facilities
- Fluorescence-activated cell sorting (FACS) capability
- Physical Containment laboratories and greenhouses
- Established expertise in protoplast isolation and transfection, CRISPR/Cas9 editing, and crop transformation and regeneration
Technology readiness level
The underlying principles are supported by validated research, including demonstrated use of RNP complexes in plant genetic engineering, fluorescence-based sorting of plant protoplasts for edit enrichment, and established protoplast isolation, transfection, and regeneration protocols for Solanaceae and Cucurbitaceae species. The approach is currently at the proof-of-concept stage, with planned validation involving co-transfection of protoplasts, FACS-based enrichment, regeneration into seedlings, and sequencing-based confirmation of the intended chromosomal inversion. The method is positioned for pilot application in crops with limited transformation infrastructure, with potential to scale as regeneration protocols are adapted across additional species.
About Flinders University
Flinders University is a comprehensive public research university based in Adelaide with a growing footprint across South Australia. Its Tonsley campus sits inside the Tonsley Innovation District, placing researchers alongside industry, government, and startups in facilities designed for co-development and demonstration. The health and medical precinct integrates the university with Flinders Medical Centre and a new Health & Medical Research Building, enabling rapid clinical collaboration and translation. Research is supported by competitive funding from the Australian Research Council, the National Health and Medical Research Council, and the Medical Research Future Fund. A dedicated research partnerships and commercialisation team and an active entrepreneurship institute help industry engage, manage IP, and launch new ventures.
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