Megabase-scale human genome rearrangement with programmable bridge recombinases

Abstract Bridge recombinases are a class of naturally occurring RNA-guided DNA recombinases. We previously demonstrated they can programmably insert, excise, and invert DNA in vitro and in bacteria. Here, we report the discovery and engineering of IS622, a simple two-component system capable of universal DNA rearrangements of the human genome. We define strategies for the optimal application of bridge systems, leveraging mechanistic insights to improve their targeting specificity. Through rational engineering of the IS622 bridge RNA and deep mutational scanning of its recombinase, we achieve up to 20% insertion efficiency into the human genome and genome-wide specificity as high as 82%. We further demonstrate intra-chromosomal inversion and excision, mobilizing up to 0.93 megabases of DNA. Finally, we provide proof-of-concept for excision of a gene regulatory region or expanded repeats relevant for the treatment of genetic diseases. Competing Interest Statement P.D.H. acknowledges outside interest as a co-founder of Stylus Medicine, Terrain Biosciences, and Monet AI, serves on the board of directors at Stylus Medicine, is a board observer at EvolutionaryScale and Terrain Biosciences, a scientific advisory board member at Arbor Biosciences and Veda Bio, and an advisor to NFDG, Varda Space, and Vial Health. M.G.D. acknowledge outside interest in Stylus Medicine. D.K. acknowledges outside interest in Shape Therapeutics. N.T.P., L.J.B., D.K., G.A.G., A.F., M.G.D., M.H., H.N., S.K., and P.D.H. are inventors on patents relating to this work. Footnotes Made minor revisions to text about bridge RNA engineering for clarity. Fixed labels in Figure 3G. Revised Figure 5 legend.