Targeted large DNA inversion in vegetables using prime editing and cre recombinase

Overview

This solution addresses one of the hardest problems in plant genome engineering: the precise inversion of very large DNA segments. Traditional homology-directed repair (HDR) cannot reliably invert fragments on the order of megabases, and prime editing alone, while capable of inserting sequences without double-strand breaks, is limited to insertions of roughly 100 bp. By combining prime editing with Cre-lox site-specific recombination, the platform enables targeted inversion of a 2 Mbp region in vegetable crops, followed by clean removal of the introduced lox sites to leave a scar-free edited genome. The approach opens new possibilities for trait development, chromosomal engineering, and synthetic biology in vegetable breeding programs.

Technical specifications

Core approach:

• Prime editing step: A binary vector delivers nCas9-RT (nickase Cas9 fused to reverse transcriptase) along with two pairs of pegRNAs to precisely insert lox sites at defined genomic locations without introducing double-strand breaks.
• Cre-mediated inversion: Once both lox sites are in place, Cre recombinase catalyzes site-specific recombination to invert the intervening 2 Mbp DNA segment.
• Scarless cleanup: A second round of prime editing removes the lox sites, restoring the genome to a near-native state suitable for regulatory review.

Key features:

• Enables inversion of DNA fragments far larger than the practical limits of HDR-based editing
• Avoids double-strand breaks during lox site insertion, reducing unintended mutations
• Modular design applicable across multiple vegetable crops with established or newly developed transformation protocols
• Combined vector system supports sequential editing and recombination in a coordinated workflow

Team capabilities:

• Vector construction for plant transformation
• Genome editing across multiple crops
• Plant transformation protocol development and optimization

Technology readiness level

The platform is at an early-to-mid stage of development. Planned validation includes establishing or adapting transformation protocols for the target vegetable crop if none are publicly available, constructing and delivering a binary vector with nCas9-RT and paired pegRNAs for lox site insertion, executing Cre-mediated recombination to achieve the 2 Mbp inversion, and applying a second round of prime editing to remove the lox sites. Each milestone requires successful plant transformation and molecular confirmation of the intended inversion and clean excision events. The approach is scientifically grounded in established prime editing and Cre-lox principles, but full proof-of-concept in a vegetable crop remains to be demonstrated.