A New Era in Genome Editing: RNA-Guided Bridge Recombinases

The site-specific insertion of gene-sized DNA fragments remains an unmet need in the field of genome editing. The IS110 family of serine recombinases was recently demonstrated to mediate programmable DNA recombination in bacteria using dual-specific RNA guides, known as bridge RNAs (bRNAs), which simultaneously recognize target and donor sites.

Recently, Martin Jinek's team at the University of Zurich, Switzerland, published a research paper entitled "Programmable genome editing in human cells using RNA-guided bridge recombinases" online in Science. This study discovered programmable genome editing in human cells using RNA-guided bridge recombinases.

In this study, researchers demonstrated that the bridge RNA-guided recombinase ISCro4, derived from Citrobacter rodentium, exhibits robust activity in human-derived cells. By engineering the bridge RNA to split into independent target-binding loops (TBL) and donor-binding loops (DBL) and applying precise RNA programming rules, they utilized ISCro4 to install kilobase-scale insertions at safe harbor loci, as well as perform programmable deletions and inversions at disease-related loci. Recombination rates for deletions and inversions in genome-integrated reporter constructs exceeded 10%. Furthermore, programmable genomic insertion of exogenous DNA was achieved with efficiencies surpassing 6%.

CRISPR-Cas genome editing has revolutionized the treatment of genetic diseases. However, effective treatment for many multi-allelic genetic disorders requires correction strategies based on the site-specific insertion of large, gene-sized DNA payloads. This remains challenging to achieve using existing first- and second-generation CRISPR genome editors. Emerging technologies aiming to address this need include Prime Medicine-assisted site-specific integrase gene editing (PASSIGE), CRISPR-associated transposons (CASTs), engineered retrotransposons, and fusions of catalytically inactive Cas9 with transposases. However, these methods face several limitations, including large coding sizes that may hinder effective cellular delivery, off-target insertions, and the creation of genomic "scars". In this context, the recently discovered bridge RNA-guided recombinases, originating from the IS110 family of bacterial insertion sequence (IS) transposable elements, represent a promising class of programmable systems for genome engineering applications.

IS110 bridge recombinases have been shown to catalyze site-specific recombination between target and donor DNA using bipartite RNA guides (referred to as bridge RNAs or seekRNAs). A bridge RNA contains two internal loops: a target-binding loop (TBL) and a donor-binding loop (DBL). Each loop contains two variable segments that base-pair with the top and bottom strands of the target and donor sites, respectively. Structural and biochemical studies of the IS621 recombinase have revealed that the recombination mechanism involves an initial nicking of the top strands of the target and donor, followed by strand exchange and ligation to create a Holliday junction intermediate, which is finally resolved through bottom-strand cleavage and ligation.

Figure 1. Structural analysis of ISCro4 to enhance activity. (Pelea O, et al., 2026)

In this work, the researchers show that the bridge recombinase ISCro4 is highly active in human cells and provide structural insights into its enhanced activity. Using either plasmid-based or all-RNA delivery, ISCro4 supports the programmable excision and inversion of several thousand base pairs and facilitates the insertion of donor DNA at genomic sites with efficiencies over 6%. Finally, the researchers evaluated the specificity and off-target activity of ISCro4. These results establish a framework for the development of bridge recombinases as next-generation editing tools that surpass current technological capabilities.