They are developing a new gene editing technique that is more precise than CRISPR.

In 2015, molecular scissors CRISPR Cas9 They were considered the greatest scientific achievement of the year. Since then, the tool has been used tirelessly in the laboratory to change or edit fragments of cellular DNA, and has also had numerous therapeutic applications. Five years later, the co-inventors of this technology received the Nobel Prize in Chemistry for their discovery.

This week, as part of a leap forward for Genetic Engineeringa team of researchers from the U.S. and Japan has discovered a new method — more precise and powerful, according to its creators — to recombine and rearrange DNA in a programmable way. The results are published today in two studies in the journal Nature.

The first of them reveals a new class of programmable biological systems. He bridging RNA is the first example of a specific guide RNA capable of simultaneously recognizing and binding to target and donor DNA sequences.

Bridge RNA is the first example of a specific guide RNA capable of simultaneously recognizing and binding to target and donor DNA sequences.

“This unique property allows us to not only insert, but also delete and programmatically invert any two pieces of DNA using one unified mechanism,” he explains to SINC. Patrick Xulead author of the paper and research fellow at the Arc Institute (Palo Alto, USA).

“Although bridged recombination represents a significant advance over the DNA and RNA cutting capabilities of previous technologies and brings us closer to the full range of genome design capabilities, it is too early to compare it with highly optimized CRISPR systems,” the expert adds.

This unique property allows us not only to insert, but also to programmatically remove and invert any two DNA fragments using a single unified mechanism.

Patrick Xu
— Arc Institute

“Certainly, our initial results in bacterial cells are promising. We have demonstrated 60% to 90% efficiency in introducing the desired gene into bacterial cells, depending on the bridging RNA used. We also achieved an insertion specificity of over 94% in the genome E. coli“, keep going.

RNA bridging systems are found in bacteria and archaeaand the researchers showed a version of this system in vitro and in bacterial cells. Its potential application in mammalian cells and genomes could benefit a wide range of organisms used in research and biotechnology.

Properties of this new method

A potential advantage of bridging RNA is that it can recombine without the need for host DNA repair machinery, which would mean more precise editing. It also has the unique ability to recognize and manipulate two DNA sequences simultaneously, opening up new capabilities that are not easily achieved with current CRISPR systems.

“We are inspired by many possible applications what we have ahead of us,” says Xu. “For example, one day entire sets of genetic variants could be modified simultaneously, allowing the study of polygenic risk factors rather than changing individual variants one at a time.”

A potential advantage of bridge RNA is that it can recombine without the need for host DNA repair mechanisms, which would mean more precise editing.

According to Xu, bridge RNA could also speed up metabolic engineering in prokaryotic biology, by engineering entire enzymatic pathways to produce valuable compounds.

Similarly, in gene and cell therapy, this mechanism will facilitate insertion of large genetic constructssuch as chimeric antigen receptors for cancer immunotherapy or missing genes for gene therapy in specific genomic regions, and improve the efficacy and safety of such treatments.

There are also many functional genomic applications, including the possibility of diseases caused by repeat expansions or genetic translocations, which can be treated by precisely cutting or reversing problematic DNA segments, allowing scientists to focus on genetic abnormalities.

Here’s how this new technology works

The bridging recombination system is derived from insertion sequence 110 (IS110) elements, one of countless types of transposable elements – or “jumping genes” – that cut and paste themselves to move within and between microbial genomes.

Mobile elements are found in all forms of life and have evolved into professional DNA manipulation machines for survival.

The IS110 elements are minimal and consist only of the gene encoding the recombinase enzyme and flanking DNA segments that have remained a mystery until now.

Xu’s lab found that when IS110 is cut from the genome, the ends of the noncoding DNA are joined together to form an RNA molecule – bridge RNA – that folds into two loops. One of these binds to the IS110 element itself, and the other to the target DNA where the element will be inserted.

Bridging RNA is the first example of a bispecific guide molecule that establishes the sequence of both target and donor DNA through base-pairing interactions.

The idea is that this new method goes beyond the DNA and RNA cutting mechanisms of CRISPR and RNA interference.

Each loop of the bridge RNA is independently programmable, allowing researchers to mix and match any target and donor DNA sequences of interest.

This means that the system can go far beyond its natural function of inserting the IS110 element, allowing the insertion of any desired genetic cargo—for example, a functional copy of a defective disease-causing gene—anywhere in the genome.

Key steps in the recombination process

The opening of Xu’s laboratory is complemented by his collaboration with the group Hiroshi Nishimasu, from the University of Tokyo, the results of which were published in a second paper in Nature.

The team used cryoelectron microscopy to determine the molecular structures of the recombinase bridge RNA complex associated with target and donor DNA, sequentially passing through the key steps of the recombination process.

While this new discovery is promising, it is important to note that CRISPR has been optimized for over a decade.

The idea is that the bridge mechanism will open up a third generation of RNA-guided systems, going beyond the DNA- and RNA-cutting mechanisms of CRISPR and RNA interference (RNAi) to offer a single programmable mechanism for DNA rearrangement.

“Until now, it was unknown how IS110 recombinase and bridging RNA work together to mediate DNA recombination,” Nishimasu told SINC. “To understand this unprecedented mechanism, we used cryo-electron microscopy to observe the atomic structure of a complex that includes IS110 recombinase, bridging RNA, donor DNA and target DNA.”

The structure revealed a detailed mechanism of how the IS110 enzyme and bridge RNA recognize donor DNA and target DNA, cut the two DNA strands, exchange and religate them, and then cut and exchange the remaining two DNA strands, completing recombination.

“This information about the IS110-bridging RNA complex can be used to design this system and create more efficient versions that can work in human cells,” the researcher notes.

References: Matthew J. Durrant and others- “Bridge RNAs direct programmed recombination of target and donor DNA.” Journal Nature, 2024 | DOI: 10.1038/s41586-024-07552-4. /// Masahiro Hiraizumi and others- “Structural mechanism of bridging RNA-driven recombination”. Journal Nature, 2024 | DOI: 10.1038/s41586-024-07570-2.