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Source: Hiraizumi, M., Perry, N.T., Durrant, M.G. et al. Structural mechanism of bridge RNA-guided recombination. Nature 630, 994–1002 (2024).

Gene editing has swiftly advanced since CRISPR was discovered, with CRISPR being developed not only to provide therapeutic gene editing treatments for humans but also for uses such as basic research, as a diagnostic tool, and for large-scale genetic screens, as well as in agricultural research to transform plant and animal traits (1). Alongside the incredible progress provided by CRISPR, scientists are seeking to discover and investigate other gene editing systems with exciting new discoveries being published that increase our understanding of the gene editing landscape and technology.

Expanding Gene Editing Capabilities

CRISPR-Cas9 has had a revolutionary impact on life sciences in a relatively short time period. Many different CRISPR-Cas9 therapies have already reached clinical trials, and in 2023, three regulatory agencies (MHRA, FDA, and EMA) approved Casgevy, the first CRISPR-Cas9 gene editing therapy to receive approval to treat patients. However, CRISPR-Cas9 does have limitations.

In hopes of expanding the capabilities of gene editing, other methods are being explored, with base editors and prime editors being two notable recent technologies. Base editing and prime editing do not create a double-stranded break, instead cutting only one strand, in contrast to CRISPR-Cas9, which works as a genetic scissors that can cut a specific DNA sequence, resulting in a double-stranded break, which is then repaired by cellular mechanisms.

Base editing converts a single target base to another; it can substitute purine bases for other purine bases or pyrimidines for pyrimidines. While base editing is highly efficient, fairly small, and works in all cell types, it is very limited in the type of error-correcting that can be done and faces the challenges of bystander editing and Cas-independent off-target deamination.

Prime editors expand the capabilities of base editing and can convert any base pair into any other, remove extra base pairs, or insert missing base pairs. They can make insertions of up to approximately 200 bases and deletions greater than 5,000 bases, making prime editing more versatile than base editing, and can potentially correct around 100 bases at once, allowing multiple changes to be made over a short region simultaneously. Prime editors are also immune to bystander editing and Cas-independent off-target deamination and seem to have very low off-target editing. However, delivery is still a hurdle. Compared to base editing, prime editors are a large, complex mechanism that requires more extensive optimization to attain high-efficiency editing.

Both base editing and prime editing have already advanced to early-stage clinical trials. Verve Therapeutics developed Verve-101, which uses base editing to treat familial hypercholesterolemia, a genetic disorder that causes dangerously high cholesterol. By changing a single letter in the DNA, Verve-101 turns off the PCSK9 gene in the liver to permanently decrease LDL cholesterol. Beam Therapeutics currently has four base editing therapeutics in Phase 1/2 trials – to treat sickle cell disease, T-cell leukemia/lymphoma, alpha-1 antitrypsin deficiency, and glycogen storage disease type 1a. Prime Medicine has begun a phase 1/2 clinical trial of PM359, using prime editing to treat chronic granulomatous disease.

These technologies provide precise small edits with the potential to treat a range of human diseases. However, Patrick Hsu, PhD, co-founder of Arc Institute, envisions a technology that can integrate and manipulate large, multi-kilobase DNA sequences. Researchers at the Arc Institute in California, in collaboration with the University of Tokyo, recently published two articles describing bridge editing, an interesting new gene editing technique that utilizes a new class of gene-editing systems capable of making precise, large-scale DNA modifications.

The Backstory: Jumping Genes

Mobile genetic elements (MGEs) are known as jumping genes, as they can cut and paste themselves throughout a genome. Autonomous transposable elements are a type of MGE that encodes all the necessary proteins for transposition, allowing them to move within a genome on their own. Insertion sequences (IS), the simplest type of autonomous transposable element found in prokaryotic genomes (2), are short DNA sequences that cause mutations and genomic rearrangements when they transpose between prokaryotic genomes. Typical IS elements are randomly inserted into new genomic loci. However, one family, the IS110 family elements, are inserted into specific DNA target sequences after they are excised as circular double-stranded DNA intermediates (2).

Investigating a New Idea

Matthew Durrant and Nicholas Perry, two members of the Hsu lab, were working on large serine recombinases (LSRs) and decided to sift through a database that Durrant had been compiling of mobile genetic elements. Perry and Durrant investigated IS110 elements, which use a recombinase to scarlessly excise themselves out of the genome and then create a circular form. IS110 elements all have a gene encoding a RuvC-like domain, and the Arc team began questioning whether a whole RNA-guided transposase system could be encoded. They focused on IS621 (an IS110 family member that is native to some strains of Escherichia coli) to investigate the potential presence of an IS110-encoded non-coding RNA (ncRNA).

The team discovered that the IS110 circular form drives the expression of a ncRNA that is otherwise unreadable, with two distinct internal binding loops reminiscent of tRNA hairpin loops or snoRNA internal loops. One loop binds to genomic insertion target DNA, while the other binds to IS110 donor DNA. The bispecific bridge RNA facilitates DNA recombination by bridging the donor and target DNA molecules through direct base-pairing interactions, thus the name bridge RNA (3). The transcribed ncRNA, purified IS621 recombinase, and target and donor DNA were combined during in vitro reconstitution experiments that proved all four components were required to form the expected recombination product (4). All of this “complex molecular logic” is encoded by bridge RNAs in a compact sequence of 150-250 nucleotides in conjunction with its single effector recombinase partner that spans 300-460 amino acids (3).

They showed that each binding loop could be programmed independently to recombine diverse DNA sequences and “that this modularity enables a generalizable mechanism for DNA rearrangement through sequence-specific insertion, inversion and excision” (3). The system could be programmed to direct excisions and inversions with notable efficiency and also to direct recombination into the genome of E. coli with high precision (4).

A second paper by Masahiro Hiraizumi, et al. was co-published with Durrant and Perry’s paper that provides the first view of this new class of RNA-guided DNA recombinase complexes. The study shows cryo-EM structures that represent the insertion step in the IS621 transposition cycle, where the circular intermediate recombines into the genomic target site, but other stages of the IS621 transposition cycle still need to be mechanistically investigated (2). It includes cryo-electron microscopy analysis of the IS621 recombinase in complex with bridge RNA, target DNA, and donor DNA, which is captured in several stages of the recombination reaction (3).

The IS621 system consists of only a single, small protein and a single non-coding RNA molecule. It accomplishes modular and programmable recognition of dDNA and tDNA and their recombination without introducing double-stranded DNA breaks (2). The team determined that IS621 recombinase catalyzes the recombination reaction consisting of top-strand cleavage and exchange, Holliday junction formation, and bottom-strand cleavage and exchange, like site-specific recombinases such as Cre. Yet, IS621 is different from all known recombinase systems by its use of a distinct domain architecture, RuvC– Tnp composite active sites, and a bRNA guide (2).

In their paper, Durrant and Perry explained that “the synaptic complex structures illustrate how two recombinase dimers associate with the target-binding loop and the donor-binding loop of bridge RNAs, coming together to form an adaptable recombination complex (ARC) with composite subunit-spanning active sites when both target and donor DNA are engaged by the ARC system.” Nicking and exchange of the top strands between the donor and target are enabled by the “elegant licensing mechanism,” and the result is a Holliday junction intermediate that is resolved by the cleavage of the bottom strands (3). According to Hiraizumi, “Our discovery of the IS110 bridge RNA illustrates a conceptually distinct way by which enzymes utilize RNA-mediated DNA recognition to manipulate nucleic acids” (2).

Durrant and Perry stated that the two studies illuminate details of the unique mode of dual-strand recognition of both target and donor DNA through programmable base-pairing interactions with the bridge RNA. They go on to claim, “Together, our genetic, mechanistic, computational and structural characterization of the bridge recombination system lays the foundation for protein and RNA engineering efforts to improve and optimize its capabilities” (3).

“Bridge editing is effectively scarless,” Patrick Hsu pointed out in a recent article. “It offers an unprecedented level of control for manipulating genomes.” Rather than simply replacing faulty genes, he thinks bridge editing could be used to completely reshape the genome of plants and animals. In another article, Hsu said, “We’re excited about the potential of this for eventually achieving chromosome-scale genome engineering, where you can do long-range insertions, deletions, and genome translocations.”

Hsu explained that the next steps include studying how to make the technique work in human cells, investigating whether the IS110 element has other functionalities that could be used in gene editing, and how to boost efficiency and precision. As for practical applications for humans, “It could provide a new way to upload genes into cell therapies for cancer and replace broken genes in inherited conditions. Plus, the ability to cut out segments of DNA could help collapse the problematic repetitive mutations responsible for neurodegenerative disorders like ALS and Huntington’s disease.”

Could Bridge Editing Expand the Editing Landscape?

Bridge editing technology is a new and very interesting continuing innovation that is evolving our understanding of the editing landscape. It has been shown to work in test tubes and bacterial cells, and it will be interesting to learn if it can work well in human cells. Like other gene editing technologies, bridge editing faces many challenges, from safety and technical hurdles to delivery and even economic challenges. In addition to discovering if it will even work in human cells, researchers must solve the problem of delivering it to various tissues. Then, once all these hurdles are overcome, the issue of cost must be addressed.

It will be interesting to discover in the coming months and years the answers to questions such as: How far can the bridge DNA go in editing? For example, can you replace kilobase-level regions of the genome or whole genes? Where would long-range insertions/deletions be used clinically? How quickly could a treatment be ready for the clinic?

Although little is known in regard to how and when it could be used clinically, the prospect and hope of a safe and effective treatment emerging from this new knowledge is exciting.

Sources:

1. Parums DV. Editorial: Genome Editing Goes Beyond CRISPR with the Emergence of ‘Bridge’ RNA Editing. Med Sci Monit. 2024 Aug 1;30:e945933. doi: 10.12659/MSM.945933. PMID: 39086277; PMCID: PMC11302237.
2. Hiraizumi, M., Perry, N.T., Durrant, M.G. et al.Structural mechanism of bridge RNA-guided recombination. Nature 630, 994–1002 (2024). https://doi.org/10.1038/s41586-024-07570-2
3. Durrant, M.G., Perry, N.T., Pai, J.J. et al.Bridge RNAs direct programmable recombination of target and donor DNA. Nature 630, 984–993 (2024). https://doi.org/10.1038/s41586-024-07552-4
4. Ertl, H. Programmable DNA rearrangements using bridge RNAs. Nat Rev Genet25, 599 (2024). https://doi.org/10.1038/s41576-024-00763-5