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 Escherichia coli. 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. Editor's summary: Precisely inserting large pieces of DNA into specific locations in the genomes of cells remains a major challenge in biotechnology and medicine. Perry et al. and Pelea et al. now introduce a genome-editing approach using a bacterial recombinase enzyme called ISCro4, guided by programmable guide RNA molecules that direct the enzyme to recognize target and donor DNA sites. These studies demonstrate that ISCro4 works efficiently in human cells, where it can remove, flip, or insert DNA segments thousands of nucleotides in length, albeit with some specificity constraints. RNA-guided recombinases could thus provide a powerful genome-editing platform to complement and extend beyond current technologies. —Di Jiang INTRODUCTION: Editing the human genome has revolutionized biology and medicine, but current tools remain constrained by the length scale of edits they can make. CRISPR systems enable precise, programmable changes, yet they are limited to relatively small DNA modifications. By contrast, recombinases can mobilize large DNA fragments but at fixed or preinstalled recognition sites, often leaving behind unwanted sequence scars. A method that combines programmable precision with the ability to insert, excise, or invert megabase-sized stretches of DNA would greatly expand our ability to study and treat human disease. RATIONALE: Bridge recombinases are a recently discovered family of enzymes that use a short "bridge RNA" (bRNA) to direct sequence-specific recombination between a pair of DNA substrates. This bispecific guidance is conceptually and mechanistically distinct from that of CRISPR nucleases and classical recombinases. We previously showed they could be used to insert DNA into bacterial genomes, suggesting the possibility of much broader applications. In this study, we asked whether bridge recombinases could be adapted to human cells, systematically optimized for activity and specificity, and used to perform programmable rearrangements across unprecedented genomic distances. RESULTS: By screening dozens of natural variants, we identified ISCro4, a bridge recombinase with robust activity in human cells. Through structural modeling, RNA engineering, and deep mutational scanning of the recombinase protein, we built an optimized system that achieved up to 20% efficiency and 82% specificity for inserting new DNA at chosen loci. Notably, an enhanced ISCro4 also performed programmable, precise, and scarless inversions approaching 1 Mb and excision events >100 kb, with no apparent distance dependency. To demomstrate therapeutic potential, we programmed ISCro4 to excise the BCL11A enhancer, a clinically validated target for sickle cell anemia therapy, and remove expanded GAA (G, guanine; A, adenine) repeats that cause Friedreich's ataxia. These proof-of-concept applications show that bridge recombinases can tackle disease-relevant genomic lesions beyond the reach of current methods. CONCLUSION: We present ISCro4 as a bridge recombinase system that establishes the first single-effector, RNA-guided technology capable of programmable megabase-scale editing of the human genome. By uniting the targeting flexibility of CRISPR with the payload mobility of recombinases, this technology enables flexible and large-scale DNA rearrangements that were previously inaccessible. Bridge recombinases can modify the genome at arbitrary new scales, ranging from single-gene insertions to megabase-sized rearrangements, which unlocks substantial potential for understanding cellular function and human disease pathology. Developing bridge recombinases for megabase-scale genome rearrangements in human cells.: (Center) Schematic of bridge recombinase, bRNA, and the recombination mechanism. (Top) Enhancing the bridge recombinase protein by deep mutational scanning (left) and bRNA by rational engineering (right). (Bottom) General applications of bridge recombinases in human cells (left) and potential therapeutic uses of bridge recombinase–mediated excision (right). TBL, target-binding loop; DBL, donor-binding loop. [ABSTRACT FROM AUTHOR]