Scientists led by Patrick Hsu at the Arc Institute in Palo Alto have unveiled a new form of gene editing that can add, remove or flip large stretches of DNA — all without the help of CRISPR.
CRISPR gene editing ushered in a new era for biology just over a decade ago. An easily reprogrammable molecule called a guide RNA, which tells the editing machinery where to cut, is key to its versatility, but CRISPR tools are best at breaking problematic genes or making small edits. Inserting whole genes or making sweeping changes to existing ones is still hard and is increasingly the focus of several new biotech startups.
Hsu’s team has found a way to overcome those challenges in a study published in Nature on Wednesday. The potential breakthrough beyond CRISPR centers around a newly discovered molecule dubbed a bridge RNA. It’s similar to a guide RNA, but recognizes two stretches of DNA at once: the target site for editing, and the new gene to be inserted. In forming that bridge, it recruits an enzyme called a recombinase to perform the actual edit.
“This allows us to specify any two DNA sequences of interest that we want to combine,” Hsu told Endpoints News in an interview. “We can insert DNA, but we can also delete it or invert it with this new system in a universal mechanism.”
Scientists have used recombinases as a research tool for decades. But the enzymes are rigidly set in their ways, only targeting specific stretches of DNA. Without a simple means to control where they make their edits, they never took off as a starting point for new medicines.
Recently, researchers began trying to control where recombinases make their edit by piggybacking the enzymes on CRISPR. Hsu’s discovery of the bridge RNA that can control recombinases could provide the simplest way yet to make the large genetic changes.
“It’s really cool. There are a lot of recombinases that we’ve known about for a long time, but one that is RNA-guided is completely new and very unexpected,” said Elizabeth Kellogg, a structural biologist and gene editing scientist at St. Jude Children’s Research Hospital. She wasn’t involved in the study.
The discovery sets the stage for competition with companies like Prime Medicine, SalioGen Therapeutics, Tessera Therapeutics, Tome Biosciences and several smaller startups working on gene insertion tools. Hsu views the technique, which he calls bridge recombination, as the start of a third wave of RNA-guided systems, following gene silencing with RNAi and gene editing with CRISPR.
“It takes us beyond what CRISPR and RNA interference do, which is fundamentally DNA and RNA cutting,” Hsu said. “And really what we want to do is not just change specific bases, but design entire genomic regions.”
The technique has only been tested in bacteria, but Hsu already has several ideas for the technology’s practical applications in humans. It could provide a new way to upload genes into cell therapies for cancer and replace broken genes in inherited conditions. And 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.
“Its quite distinct from CRISPR,” Samuel Sternberg, a gene editing scientist at Columbia University who wasn’t involved in the study, told Endpoints. “But there’s still going to be a lot of work to do to really determine whether this technology will open up new doors in other cells, or whether it will be just a really remarkable piece of biological research.”
Arc Institute scientists (L-R) Patrick Hsu, Nick Perry and Matt Durrants (Photographer: Ray Rudolph)
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From ‘jumping genes’ to gene editor
The new tool came from bacterial jumping genes, named for their ability to cut and paste themselves throughout a genome. They sometimes encode helpful gifts to their hosts, such as antibiotic resistance, in exchange for hitchhiking. Hsu’s lab was interested in one of the most minimal insertion sequences, called IS110.
IS110 was discovered nearly 40 years ago. It’s a stretch of DNA encoding an enzyme that cuts the jumping gene out of its home and stitches it into its new digs. In between, the two ends of the excised gene loop around to form a circle, triggering the host to make more copies of itself.
When Hsu’s lab made these circles and put them in bacteria, they noticed something unusual: a hidden message within the two flanks of the jumping gene that was only readable when they formed a circle. It was instructions for making an RNA molecule. No one had noticed it before.
Hsu was well primed to the potential importance of this mysterious RNA, thanks to his role developing gene editing tools as a graduate student in CRISPR pioneer Feng Zhang’s lab at the Broad Institute. The ease of making new guide RNA molecules to tell CRISPR where to go is what makes the tool so versatile. Hsu wondered: Could this be a guide RNA for a jumping gene?
There were two smoking guns. Stretches of DNA in the bacterial genome and in the jumping gene mirrored the code of the RNA, suggesting that they paired up — much like a guide RNA would, only this one recognized two parts of DNA instead of one. “We named this the bridge RNA, inspired by its natural role in bridging target and donor DNA,” Hsu said.
The lab created synthetic bridge RNA and showed that it could tweak its code to change where the jumping genes landed (the target) and which jumping gene got inserted (the donor) in test tubes and bacteria. In addition to inserting genes, the lab also showed the bridge RNA could be programmed to cut out or invert genes.
“It’s very exciting, but a tool like this is going to have to be further engineered for any sort of clinical application,” Kellogg said. “There’s a range for the efficiency of targeted integrations. Sometimes it works really well. Sometimes it doesn’t.”
“Furthermore, we don’t know if this works in human cells, and it’s not necessarily obvious that these tools would translate,” Kellogg added. “That also needs to be explored.”
The paper focused on one kind of IS110 element, but there are actually many versions of that jumping gene found in nature. Hsu said his lab is exploring other jumping genes and engineering them for use in eukaryotic and mammalian cells, not just bacteria.
Hsu is involved in several biotech companies, including the stealth startup Stylus Medicine, which has an undisclosed amount of funding from RA Ventures and Khosla Ventures, according to its website. Hsu wouldn’t confirm whether bridge recombination was a focus of that company, but its website mentions “a novel class of enzymes to enable the insertion of any-length genetic sequences” — a description that meshes with Hsu’s new technology.