Programmable genome editing in human cells using RNA-guided bridge recombinases

Site-specific insertion of gene-sized DNA fragments remains an unmet need in the genome editing field. IS110-family serine recombinases have recently been shown to mediate programmable DNA recombination in bacteria using a bispecific RNA guide (bridge RNA) that simultaneously recognizes target and donor sites. Here, we show that the bridge recombinase ISCro4 is highly active in human cells, and provide structural insights into its enhanced activity. Using plasmid- or all-RNA-based delivery, ISCro4 supports programmable multi-kilobase exisions and inversions, and facilitates donor DNA insertion at genomic sites with efficiencies exceeding 6%. Finally, we assess ISCro4 specificity and off-target activity. These results establish a framework for the development of bridge recombinases as next-generation tools for editing modalities that are beyond the capabilities of current technologies.