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 field of genome editing. IS110-family serine recombinases have recently been shown to mediate programmable DNA recombination in bacteria by using a bispecific RNA guide (bridge RNA) that simultaneously recognizes target and donor sites. In this work, we have shown that the bridge recombinase ISCro4 is highly active in human cells and provided structural insights into its enhanced activity. Using plasmid- or all-RNA–based delivery, ISCro4 supports programmable multikilobase excisions and inversions and facilitates donor DNA insertion at genomic sites with efficiencies that exceed 6%. Last, we assessed 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. 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: Precise manipulation of large genomic segments remains a major unmet goal in genome editing. Although CRISPR-Cas nucleases, base editors, and prime editors enable efficient gene disruption or correction of small mutations, they remain limited in their ability to support the site-specific insertion of large, gene-sized DNA fragments. Such precise integrations would enable correction of multiallelic genetic disorders that cannot be addressed with small-scale edits. Moreover, the ability to induce large deletions and inversions in a scar-free manner would permit both modeling and correction of structural variants. Current strategies based on homology-directed repair, integrases, or transposons are hampered by low efficiencies, genomic scarring, off-target insertions, and large coding sizes that complicate delivery. These limitations highlight the need for compact, programmable systems capable of mediating seamless genomic rearrangements in mammalian cells. RATIONALE: Bridge recombinases from the IS110 family catalyze site-specific recombination between donor and target DNAs as guided by a bipartite bridge RNA guide composed of a target-binding loop (TBL) and a donor-binding loop (DBL). Although the prototypical IS621 enzyme can be reprogrammed to mediate deletions, inversions, and insertions in bacteria, its activity in human cells is low. This study aimed to identify RNA-guided recombinases capable of mediating programmable recombination in human cells that would enable their technological translation. RESULTS: Initial screening of a panel of candidate IS110-family bridge recombinases revealed that ISCro4 from Citrobacter rodentium displayed robust activity in human-derived cultured cells. A cryo–electron microscopy structure of the ISCro4 synaptic complex revealed that the enhanced activity of ISCro4 is in part due to additional molecular contacts with the bound nucleic acids. The in cellulo activity of ISCro4 could be increased nearly twofold by splitting the bridge RNA into independent TBL and DBL guide RNAs. Using optimized plasmid-based delivery, or an all-RNA approach that combines ISCro4 mRNA with chemically modified TBL and DBL RNAs, ISCro4 mediated chromosomal deletions and inversions with efficiencies that exceed 10% in genome-integrated reporter cell lines. Furthermore, ISCro4 could be programmed to enable donor DNA insertion at endogenous genomic sites with efficiencies greater than 6%, as well as excisions and inversions at disease-relevant loci. Comprehensive off-target profiling of ISCro4 revealed recombination at genomic sites containing up to two mismatches, as well as variable levels of unintended DBL-DBL and TBL-TBL recombination. CONCLUSION: This study establishes ISCro4 as a compact, RNA-guided recombinase capable of scar-free genomic deletions, inversions, and insertions. ISCro4 thus presents a versatile molecular platform that extends programmable genome editing beyond CRISPR-based technologies. Although the genomic specificity of ISCro4 is intrinsically constrained by the 14-nucleotide size of its donor and target sites, stringent selection of unique genomic target sites coupled with genome-orthogonal donors presents a first-line strategy to minimize off-target recombination activity for programmable genomic insertions. Future technology development efforts, focused on large-scale guide RNA profiling and computational prediction, coupled with improved delivery methods and molecular engineering of recombinases and their guide RNA scaffolds, will yield improved systems with enhanced precision and broader applicability for biotechnological and therapeutic applications. Bridge recombinase ISCro4 mediates RNA-programmed DNA recombination.: ISCro4 is directed by a bridge RNA with two corresponding loops: a TBL and a DBL that guide recombination between 14-nucleotide target and donor DNA sites. This mechanism enables the introduction of genomic deletions, inversions, and targeted insertions. [ABSTRACT FROM AUTHOR]