○Masahiro Hiraizumi1 Hiroshi Nishimasu11 Dept. of Chem. & Biotech., Grad. Sch. of Eng., The Univ. of Tokyo.PurposeBridge recombinases are recently discovered RNA-guided DNA recombination systems from IS110 family transposons (Durrant et al., Nature, 2024). They consist of a recombinase enzyme and a non-coding bridge RNA (bRNA) that together catalyze site-specific DNA rearrangements. We aim to elucidate the structural mechanism by which a bispecific bRNA directs recombination between a target DNA and a donor DNA, and to harness this mechanism for programmable genome editing applications such as precise excision of pathogenic DNA elements.MethodsWe combined biochemical assays with cryo-electron microscopy (cryo-EM) structure determination to characterize the IS110 bridge recombination complex (Hiraizumi et al., Nature, 2024). Using reprogrammed bridge RNA loops and engineered recombinase variants, we tested DNA targeting and recombination in both bacterial and human cell systems (Perry et al., Biorxiv, 2025).ResultStructural analysis revealed that bridge RNA comprises two internal loops, each independently recognizing target or donor DNA, thus linking both DNAs to recombinase dimers. Cryo-EM structures showed a synaptic complex wherein two recombinase dimers are bridged by the RNA, creating a composite active site. This arrangement positions catalytic residues for sequential DNA cleavage, strand exchange through a Holliday junction intermediate, and ligation. Consequently, precise DNA insertions, excisions, or inversions occur at defined genomic sites.DiscussionOur findings reveal RNA-guided DNA recombination system that expands the genome editing toolkit beyond CRISPR-based nucleases. The modularity of the bridge RNA loops allows retargeting to diverse sequences, and strategic engineering has achieved high-efficiency (up to ~ 20%) insertions and megabase-scale genomic rearrangements in human cells. This work establishes the structural basis for RNA-guided DNA recombinases and highlights their potential for programmable genome engineering and therapeutic genome rearrangements. Bridge RNAs direct programmable recombination of target and donor DNA 34P 004
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