CRISPR/Cas-mediated targeted genomic inversion for vegetable crop improvement

Overview

This solution offers a genome editing approach that uses CRISPR/Cas nucleases with paired guide RNAs to create targeted chromosomal inversions of up to 2 Mbp in vegetable crops. By inducing double-strand breaks at two distant sites on the same chromosome, the platform enables the repositioning of commercially important genes into proximity, unlocking new trait combinations for plant breeding programs. The technology addresses a key limitation in conventional breeding by allowing precise structural rearrangement of the genome rather than relying on naturally occurring inversions.

Technical specifications

• Paired guide RNA design: Two guide RNAs target proximal and distal genomic positions on the same chromosome to create flanking double-strand breaks
• Nuclease delivery options: RNP complexes, DNA vectors, or protein delivery via biolistics, Agrobacterium, or engineered Agrobacterium expressing a type III secretion system
• Vector construction: Guides and Cas nucleases cloned with appropriate promoters and terminators for vegetable species
• Guide screening: Efficient guides identified through protoplast delivery and genotyping prior to whole-plant experiments
• Inversion detection: PCR and sequencing-based confirmation of inversion events
• Regeneration pipeline: Transformed cells selected, tissues regenerated, and plants rooted in controlled growth chamber conditions
• Inheritance validation: Confirmed stable inheritance of inversions to subsequent generations, as demonstrated in prior rice and Nicotiana benthamiana studies
• Prior validation data: Achieved 62.9% mutation efficiency in rice with two PAM-out guide RNAs, including confirmed inversions of 108 bp and 269 bp; similar inversion efficiencies observed in Nicotiana benthamiana using combinatorial guide RNA delivery

Technology readiness level

The approach has been validated in model and crop species including rice, Arabidopsis, corn, and Nicotiana benthamiana, with demonstrated inversion events up to 75.4 Mbp in published studies. Prior work by the research team has confirmed that induced inversions are stably inherited across generations. The technology is positioned at TRL 4–5, having moved beyond proof-of-concept in model systems toward application in vegetable species. Future validation will involve receiving target vegetable seeds from industry partners, confirming sequence similarity at target sites, regenerating edited tissues, and conducting greenhouse-based trait evaluations to advance toward commercial readiness.