Microfluidic screening platform for enrichment of rare DNA insertion events in plant cells

Overview

A microfluidic cell-screening platform designed to recover rare DNA insertion events from plant-cell populations without relying on permanent selectable markers. Current DNA-insertion methods, including prime editing with extended donors, recombinases, transposases, and staged HDR–recombinase strategies, often achieve efficiencies below 5% and remain target-dependent. Conventional Agrobacterium transformation compensates through selectable markers, but this approach is poorly suited to next-generation trait development where edits could occur naturally.

This platform bridges that gap by combining protoplast transfection with microfluidic, fluorescence-based cell sorting and enrichment. Because enrichment is independent of the insertion mechanism, the platform can support a wide range of DNA-insertion technologies. The workflow preserves cell viability and regenerative capacity, avoiding the losses associated with conventional FACS.

Technical specifications

• Microfluidic fluorescence-based sorting of plant protoplasts, designed to be gentle enough to maintain regenerative capacity
• Insertion-mechanism agnostic: compatible with prime editing, recombinases, transposases, HDR-based methods, and other DNA-insertion approaches
• Marker-free enrichment: recovers rare insertion events without permanent selectable markers, making it well-suited to natural or non-transgenic trait changes
• Validated regeneration after sorting demonstrated in commercial potato and tomato lines
• Proof-of-enrichment demonstrated: sorting protoplasts transfected with CRISPR-Cas9::GFP RNPs for high GFP signal increased gene-editing knockout efficiency by 20%
• Intellectual property: workflow protected by two patents (one granted, one pending)
• Planned benchmarking: comparative testing across GFP-traceable donors, CRISPR-Cas, recombinase, transposase systems, and delivery plasmids, with optimisation of transfection, sorting, and recovery timing, followed by NGS validation of insertion efficiency and fidelity

Technology readiness level

The core microfluidic sorting and regeneration workflow has been demonstrated in commercial potato and tomato lines, with a 20% improvement in gene-editing knockout efficiency shown as proof of enrichment. Increased insertion efficiency has not yet been robustly demonstrated due to resource constraints. A planned six-month programme of repeated design-build-test cycles, each lasting approximately 10 days, will benchmark enrichment across multiple DNA-insertion methods and target loci to establish a robust proof-of-concept workflow with quantified gains in true insertion event recovery.