DNA-free genome editing method for gene inversion in vegetable crops

Overview

This offering presents a novel DNA-free genome editing method for inducing targeted gene inversions in vegetable crops. The technology leverages the delivery of ribonucleoprotein complexes (RNPs) into plant cells using a proprietary laser-mediated transformation method, with biolistic transformation as a backup. By introducing double-strand DNA breaks at two positions flanking a gene of interest, the approach exploits spontaneous DNA recombination events to generate chromosomal inversions—a technique known as chromosome engineering. This method enables precise genetic modifications without introducing foreign DNA into the host genome, making it particularly attractive for breeding programs and trait development in vegetable crops.

Technical specifications

Key features:

• DNA-free editing: Uses RNPs rather than plasmid DNA, eliminating the risk of foreign DNA integration in the host plant genome
• Laser-mediated transformation: A gentle, proprietary delivery method that efficiently shuttles RNPs into single cells of leaf explants without impairing plant regeneration
• High mutation efficiency: Validated in tobacco knockout studies with consistently high gene editing rates
• Chromosome engineering capability: Induces inversions of large DNA fragments (up to 75.5 Mb), as previously demonstrated in Arabidopsis and maize
• Biolistic backup: Alternative transformation method available to ensure experimental flexibility
• Comprehensive validation pipeline: Includes high-throughput PCR screening, deep sequencing characterization, greenhouse crossing, and homozygous mutant identification

How it works:

The process involves designing RNPs that target two regions flanking the gene of interest, producing a double cut in the genome. Spontaneous recombination events then lead to the desired inversion at low frequency. Transformed tissue explants are cultured to regenerate plantlets, which are subsequently screened and crossed to obtain homozygous mutants.

Technology readiness level

The laser-mediated DNA-free gene editing method has been validated in tobacco for gene knockout applications. The chromosome engineering concept—inducing large DNA fragment inversions via double-strand breaks—has been previously validated by other research groups in Arabidopsis and maize. The current proposal aims to extend this approach to vegetable crops through four experimental phases: (1) establishing plant regeneration protocols, (2) performing RNP transformation and tissue culture, (3) characterizing mutants via PCR and deep sequencing, and (4) growing and selecting homozygous mutants in greenhouses. Final mutant plants will be transferred to the sponsoring company for further evaluation.