Megabase-scale human genome rearrangement with programmable bridge recombinases

Bridge recombinases are naturally occurring RNA-guided DNA recombinases that we previously demonstrated can programmably insert, excise, and invert DNA in vitro and in <i>Escherichia coli</i>. In this study, we report the discovery and engineering of the bridge recombinase ortholog ISCro4 for universal rearrangements of the human genome. We defined strategies for the optimal application of bridge systems, leveraging mechanistic insights to improve their targeting specificity. Through rational engineering of the ISCro4 bridge RNA and deep mutational scanning of its recombinase, we achieved up to 20% insertion efficiency into the human genome and genome-wide specificity as high as 82%. We further demonstrated intrachromosomal inversion and excision, mobilizing up to 0.93 megabases of DNA. Lastly, we provided proof of concept for plasmid-based excision of disease-relevant gene regulatory regions or repeat expansions.