Precise manipulation of large genomic fragments is a critical need in crop breeding and synthetic biology. In crop genomes, superior traits are often associated with structural variations. However, existing large-fragment editing tools such as CRISPR-Cas systems and site-specific recombinases have limitations.

In 2024, the development of the bridge RNA-guided IS110-family recombinase system provides a new way for programmable, double-strand break (DSB)-free, scarless large-fragment genome editing, but its application in plants has not been demonstrated.

In a study published in Trends in Biotechnology, a team led by Prof. GAO Caixia from the Institute of Genetics and Developmental Biology (IGDB) of the Chinese Academy of Sciences developed a bridge RNA-guided recombinase system that enables programmable, scarless large-fragment genome editing across both plant and mammalian cells.

Researchers evaluated two IS110-family recombinases, IS621 and ISCro4, in plant cells, and selected ISCro4 as the enzymatic core for its robust activity across insertion, deletion, and inversion editing. They engineered bRNA scaffold to stabilize the target-binding loop stem-loop structure and optimize the donor-binding loop conformation, improving editing efficiency by up to 15.3-fold over wild-type bRNA.

Then, researchers engineered ISCro4 recombinase by using AI-assisted directed evolution platform, AiCE. They identified two key mutations, E220A and D164E, which stabilize recombinase-bRNA-DNA ternary complex. Notably, co-optimization of bRNA and recombinase exhibited strong synergy: double mutant Rm7 paired with optimized bRNA_mT6D16 achieved a 29.8-fold overall improvement in activity, reaching 9.4% editing efficiency in rice protoplasts.

In regenerated rice plants, the new system mediated precise deletions and inversions of 27-315 kb fragments with editing efficiencies up to 23.9%. Through a 315-kb genomic inversion, the rewiring of an endogenous promoter to OsHIS1 gene upregulated its expression 15.1-fold and conferred enhanced herbicide resistance.

Whole-genome sequencing of the edited plants confirmed all intended rearrangements with no detectable off-target structural variations, demonstrating the high specificity of the system.

Moreover, by exploiting the compact size of ISCro4 (<350 amino acids), researchers packaged recombinase together with bRNA expression cassette into a single adeno-associated virus vector, and achieved 31-kb inversions and 30-kb deletions in mammalian cells.