Visualization of the bridge recombinase mechanism. Credit: Visual Science
Scientists at the Arc Institute have discovered the bridge recombinase mechanism, a revolutionary tool that enables fully programmable DNA rearrangements.
Their findings, recently described in a report, Nature publication, is the first DNA recombinase that contains a non-coding RNA for sequence-specific selection of target and donor DNA molecules. This bridge RNA is programmable, allowing the user to specify any desired genomic target sequence and donor DNA molecule to be inserted.
The research was conducted in collaboration with the laboratories of Silvana Konermann, principal investigator at the Arc Institute and assistant professor of biochemistry at Stanford University, and Hiroshi Nishimasu, professor of structural biology at the University of Tokyo.
Visualization of the bridge recombinase mechanism with the donor and target binding loops. Credit: Visual Science
A new era of genetic programming
“The bridge RNA system is a fundamentally new mechanism for biological programming,” said Dr. Patrick Hsu, lead author of the study and an Arc Institute Core Investigator and University of California, Berkeley Assistant Professor of Bioengineering. “Bridge recombination can universally modify genetic material through sequence-specific insertion, excision, inversion, and more, enabling a text editor for the living genome that goes beyond CRISPR.”
The bridge recombination system originates from insertion sequence 110 (IS110) elements, one of numerous types of transposable elements – or “jumping genes” – that cut and paste themselves to move within and between microbial genomes. Transposable elements are found in all life forms and have evolved into professional DNA-manipulating machines to survive. The IS110 elements are very minimal, consisting only of a gene that encodes the recombinase enzyme, plus flanking DNA segments that have remained a mystery until now.
Visualization of the bridge recombinase mechanism with emphasis on the transposon DNA and genomic target site. Credit: Visual Science
Advanced mechanism of Bridge RNA
The Hsu lab found that when IS110 removes itself from a genome, the noncoding DNA ends join together to produce an RNA molecule—the bridge RNA—that folds into two loops. One loop binds to the IS110 element itself, while the other loop binds to the target DNA where the element will be inserted. The bridge RNA is the first example of a bispecific guide molecule, specifying the sequence of both target and donor DNA through base-pairing interactions.
A team of researchers from the Arc Institute has discovered the bridge recombinase mechanism, a precise and powerful tool to recombine and rearrange DNA in a programmable way. Going far beyond programmable genetic scissors like CRISPR, the bridge recombinase mechanism allows scientists to specify not only the target DNA to be modified, but also the donor material to be recognized, allowing them to insert new, functional genetic material, excise defective DNA, or invert any two sequences of interest. Learn more in this short video visualizing key aspects of the bridge recombination mechanism. Credit: Visual Science
Each loop of the bridge RNA is independently programmable, allowing researchers to mix and match any desired target and donor DNA sequences of interest. This means that the system can go far beyond its natural role of inserting the IS110 element itself, and instead enable the insertion of any desired genetic payload, such as a functional copy of a defective, disease-causing gene, into any genomic location. In this work, the team demonstrated greater than 60% insertion efficiency of a desired gene into E. coli with a specificity of more than 94% for the correct genomic location.
“These programmable bridge RNAs distinguish IS110 from other known recombinases, which lack an RNA component and cannot be programmed,” said co-lead author Nick Perry, a bioengineering doctoral student at UC Berkeley. “It’s as if the bridge RNA is a universal power adapter that makes IS110 compatible with any electrical outlet.”
Patrick Hsu, Nick Perry and Matt Durrant discuss the recently discovered bridge recombinase mechanism. Credit: Ray Rudolph
Collaborative research and future implications
The Hsu lab’s discovery is complemented by their collaboration with the lab of Dr. Hiroshi Nishimasu at the University of Tokyo, also published June 26 in NatureThe Nishimasu lab used cryo-electron microscopy to determine the molecular structures of the recombinase-bridge-RNA complex bound to target and donor DNA, sequentially tracing the key steps of the recombination process.
Januka Athukoralage, Nicholas Perry, Silvana Konermann, Matthew Durrant, Patrick Hsu, James Pai and Aditya Jangid. Credit: Ray Rudolph
With further exploration and development, the bridge mechanism promises to usher in a third generation of RNA-guided systems, which will go beyond the DNA and RNA cutting mechanisms of CRISPR and RNA interference (RNAi) to provide a unified mechanism for programmable DNA rearrangements. The bridge recombinase is crucial for the further development of the bridge recombination system for mammalian genome design and connects both DNA strands without releasing cut DNA fragments, thereby circumventing a major limitation of current advanced genome editing technologies.
“The bridge recombination mechanism solves some of the most fundamental challenges faced by other genome editing approaches,” said study leader Matthew Durrant, a senior scientist at Arc. “The ability to programmatically rearrange any two DNA molecules opens the door to breakthroughs in genome design.”
References:
“Bridge RNAs direct programmable recombination of target and donor DNA” by Matthew G. Durrant, Nicholas T. Perry, James J. Pai, Aditya R. Jangid, Januka S. Athukoralage, Masahiro Hiraizumi, John P. McSpedon, April Pawluk, Hiroshi Nishimasu, Silvana Konermann, and Patrick D. Hsu, June 26, 2024, Nature.
DOI file: 10.1038/s41586-024-07552-4
“Structural mechanism of bridge RNA-guided recombination” by Masahiro Hiraizumi, Nicholas T. Perry, Matthew G. Durrant, Teppei Soma, Naoto Nagahata, Sae Okazaki, Januka S. Athukoralage, Yukari Isayama, James J. Pai, April Pawluk, Silvana Konermann, Keitaro Yamashita, Patrick D. Hsu and Hiroshi Nishimasu, June 26, 2024, Nature.
DOI file: 10.1038/s41586-024-07570-2