Engineering RNA-guided bridge recombinases for precise large-scale genome editing.

Abstract

Precise manipulation of large DNA fragments in eukaryotic genomes remains limited by the low efficiency and delivery constraints of current multicomponent editing systems. In this study, we engineered an RNA-guided bridge recombinase system through rational mutagenesis and AI-assisted directed evolution, enabling programmable chromosomal rearrangements in both plant and mammalian cells and achieving up to a 29.8-fold increase in activity. In plants, the optimized system mediated precise deletions, insertions, and inversions from 1.8- to 315-kb DNA fragments, with stable editing efficiencies of up to 23.9% in regenerated rice plants. We further generated herbicide-resistant rice through a 315-kb chromosomal inversion that rewired endogenous promoter activity. In mammalian cells, the compact ISCro4 recombinase system was delivered using a single adeno-associated virus vector, thereby supporting efficient genome editing. Together, these results establish bridge RNA-guided recombinases as a versatile platform for programmable chromosome-scale genome engineering, with broad potential for precision breeding and gene therapy.