For decades, gene-editing science has been limited to making small, precise edits to human DNA, akin to correcting typos in the genetic code. Arc Institute researchers are changing that paradigm with the development of a universal gene editing system that allows for cutting and pasting of entire genomic paragraphs, rearranging whole chapters, and even restructuring entire passages of the genomic manuscript.
In a published paper in Science, the research team, headed by Patrick Hsu, PhD, an Arc Institute core investigator and University of California, Berkeley bioengineering faculty member, demonstrated how bridge recombinase technology can be applied to human cells. The advancement allows scientists to manipulate large genomic regions, testing up to a million base pairs in length, by inserting new genes, deleting entire gene clusters, or inverting regulatory sequences.
“Bridge recombinases could transform how we create genetic therapies by offering one versatile medicine per patient population instead of thousands of individual treatments,” said Hsu. “With the ability to move and reshape entire genetic regions, we can engineer biology at the scale that evolution operates upon and apply those capabilities to solving complex diseases.” Hsu is senior author of the team’s report, which is titled “Megabase-scale human genome rearrangement with programmable bridge recombinases”, in which the team stated, “Our optimized approach for universal chromosomal DNA rearrangements achieves as high as 20% insertion efficiency and mobilizes genomic DNA at the megabase scale.”
Bridge recombinases were discovered from parasitic mobile genetic elements that hijack bacterial genomes for their own survival. Described in 2024 by Hsu and colleagues in Nature, these elements encode both a new class of structured guide RNA, which they named a bridge RNA (bRNA), and a recombinase enzyme that rearranges DNA. Hsu and colleagues repurposed this natural system by reprogramming the bridge RNA to target new DNA sequences, creating the foundation for a new type of precise gene editing tool. “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,” the authors explained. “The bispecific and programmable mechanism of bridge recombinases naturally circumvents many of the fundamental engineering challenges in the recombinase field.”
The team’s initial study provided proof-of-concept for using bridge recombinases in programmable modification of prokaryotic genomes. In their newly released report in Science the team described the discovery and engineering of a bridge recombinase with high activity in human cells. Starting with 72 different natural bridge recombinase systems isolated from bacteria, they found that about 25% showed some activity in human cells, but most were barely detectable. Only one system, called ISCro4, showed enough measurable activity to enable further optimization.
“In this study, we present ISCro4, the first bridge recombinase for programmable insertion, inversion, and excision of the human genome,” they reported. The team enhanced its activity by rational engineering of the bRNA via point mutations and scaffold modifications.
The scientists then systematically improved both the protein and its RNA guide components, testing thousands of variations until they achieved 20% efficiency for DNA insertions and 82% specificity for hitting intended targets in the human genome.
While CRISPR uses a single guide RNA to target one DNA location, bridge RNAs are unique because they can simultaneously recognize two different DNA targets through distinct binding loops. This dual recognition enables the system to perform coordinated rearrangements such as bringing together distant chromosomal regions to excise genetic material or flipping existing sequences in reverse orientation. The system acts as molecular scaffolding that holds two DNA sites together while the recombinase enzyme performs the rearrangement reaction.
As a proof-of-concept, the researchers created artificial DNA constructs containing the same toxic repeat sequences that cause progressive neuromuscular decline in Friedreich’s ataxia patients. While healthy individuals carry fewer than 10 sequential copies of a three-letter DNA sequence, people with the disorder can harbor up to 1,700 copies, which interferes with normal gene function. Hsu and colleagues demonstrated that the engineered ISCro4 successfully removed these repeats from the artificial constructs, in some cases eliminating over 80% of the expanded sequences.
“Since disease severity correlates with repeat length, any amount of excision, whether it’s a perfectly healthy genotype or not, has the potential to improve patient symptoms,” said lead author Nicholas Perry, PhD, an Arc scientist in the Hsu Lab who also conducted this research as a UC Berkeley PhD student. “Bridge recombinases could apply to any heritable disease that result from expansions, and because we only need to deliver RNA molecules rather than proteins or DNA to make it work inside human cells, the approach could be much simpler to implement and scale.”
The team also demonstrated that bridge recombinases could replicate existing therapeutic approaches by successfully removing the BCL11A enhancer, the same target disrupted in an FDA-approved sickle cell anemia treatment. And because bridge recombinases can move massive amounts of DNA, the technology could also help model the large-scale genomic rearrangements associated with cancers.
“Beyond gene insertion, we used ISCro4 for programmable, precise, and scarless genome rearrangements, inverting up to 0.92 Mb and excising up to 0.13 Mb with no apparent distance dependency,” the authors wrote in summary. “… we demonstrated therapeutic proof-of-concept with excision of the BCL11A enhancer for sickle cell anemia and of repeat sequences found in Friedreich’s ataxia.”
The investigators are now working to expand the platform’s capabilities, including testing bridge recombinases in clinically relevant immune cells and stem cells, developing therapeutic delivery methods, and engineering variants that can handle DNA segments larger than a million base pairs. They also plan to explore applications in plant genetics and synthetic biology. “Bridge recombinases can modify the genome at arbitrary new scales, ranging from single gene insertions to megabase-sized re-arrangements, which unlocks significant potential for under-standing cellular function and human disease pathology,” they further stated.
Perry concluded, “What’s different about our new paper is not only are we able to show insertion into the human genome but we’re also showing quite efficiently the excision and inversion of genomic sequences in a programmable way. The applications of this platform are particularly exciting and could apply broadly across many kinds of scientific projects.”
In their paper the team suggested, “We expect future efforts for improving bridge recombinases to focus on further improvement of targeting efficiency and specificity, delivery of megabase-scale DNA payloads, and development of therapeutic delivery formulations, including RNA-only delivery for genomic rearrangements such as repeat excisions. Additionally, recombinase-bRNA electroporation may prove effective, similar to previous efforts for delivery of CRISPR effectors and guide RNAs.”
