Bridge RNA as a Promising Genome Editing Platform

The advent of CRISPR-Cas has transformed genome editing, but it remains limited by delivery challenges and off-target effects, leading to the development of improved tools, such as base and prime editors. Still, many genetic diseases—such as cystic fibrosis and retinitis pigmentosa—stem from diverse mutations in a single gene and require the site-specific insertion of large DNA fragments, which is beyond the reach of current methods.

To address this gap, researchers at the University of Zurich investigated whether bridge RNA-guided recombinases, which can cause precise DNA rearrangement in vitro and in bacteria, could be implemented in mammalian cells. In a new Science study, the team demonstrated that the bridge recombinase ISCro4 worked in human cell lines and carried out corrective genome editing at clinically relevant genomic loci.¹ These findings underscore the potential of bridge recombinases as a molecular platform for developing the next generation of genome engineering tools.

Bridge RNA-guided recombinases originate from the IS110 family of bacterial insertion sequence transposable elements. These mobile elements can orchestrate large genomic rearrangements; they express a noncoding RNA—the bridge RNA—that contains two binding loops that can recognize target and donor DNA. These loops can be engineered to reprogram the recombinase IS621 to invert, excise, or insert large DNA sequences at specific regions.²

Here, the researchers first transfected a human embryonic kidney (HEK293T) cell line with IS621; however, using flow cytometry, they found that these cells lowly expressed IS621 recombinase

So, they assessed other IS110-family bridge recombinases and found that ISCro4, an ortholog of IS621, has high activity of RNA-guided recombination in mammalian cells.³ It effectively deleted gene-sized fragments from plasmids in HEK293T cells.

To understand the source of the observed efficiency differences, the researchers focused on structural variations formed during this process within the complex. They identified 39 residues that differed between the bridge recombinases. Of these, ISCro4 had more serines, which appeared to form additional hydrogen bonds. The increased interactions with these nucleic acids likely contributed to ISCro4’s enhanced activity, along with other subtle amino acid substitutions in the hydrophobic core and solvent-exposed regions of the enzyme’s surface.

The researchers then tested whether ISCro4 could mediate inversions and insertions in HEK293T and K562, a lymphoblast cell line. They found that ISCro4 mediated gene-scale rearrangements at varying efficiencies across the two cell types, achieving up to 75 percent inversion in HEK293T cells and 36 percent deletion in K562 cells, for instance. The findings suggest that the approach is likely generalizable across mammalian cells.

To tackle genetic diseases that demand correction of large DNA segments, the researchers put ISCro4 to the test at multiple disease-relevant genomic loci. ISCro4 efficiently edited genes associated with cystic fibrosis, sickle cell disease, and beta-thalassemia, highlighting its potential for therapeutic applications.

While further optimization is required, the researchers believe that ISCro4 and bridge recombinases in general serve as a promising system for tackling challenges around CRISPR-based technologies.