years can now be run in weeks, but now researchers need the tools to detect edits just as quickly and easily, Corn says.
(IGI’s executive director is Jennifer Doudna, a UC Berkeley professor whose pioneering work on CRISPR/Cas9 helped turn it into a genome-editing technology. I wrote about her role in an ongoing patent battle here.)
As carefully engineered as the scissors and the guide might be, though, therapeutic uses could end up with millions of copies going into millions of cells per patient. Mistakes will be made, as a politician might say.
But how many? And where in the DNA will they occur? And how are they different from the constant DNA cuts and mutations happening constantly in our cells, which for the most part our cells know how to deal with?
Especially with CRISPR/Cas9, there is a huge gap between the ability to produce new guides (and hit new targets) and the ability to see if—and where—the scissors went awry. “The most pressing need is to know how CRISPR/Cas9 works,” says Corn. “It’s only been around three years now.”
Knowing where and how often gene-editing technology is making those cuts will give therapeutics developers more data points to consider as they move a product toward clinical trials. Keith Joung, a researcher at Massachusetts General Hospital who coauthored one of the new studies, likens the knowledge to the preclinical toxicology tests that developers of traditional chemical drugs use. (Joung is also a scientific founder of Editas Medicine of Cambridge, MA, one of three venture backed startups competing to develop therapies that use CRISPR/Cas9.)
How can scientists find the wayward cuts? In their study, Joung, Shengdar Tsai (a postdoc in Joung’s MGH lab), and colleagues took advantage of a well-known trait of cell repair. When a cell fixes broken strands of DNA, other material nearby can get incorporated into the fix. The researchers introduced tiny oligonucleotide tags into cells at high concentrations, which were taken up into the repair sites. Those tags could then be tracked and counted. “The number of times you see a tag ‘hop’ into a particular place would correlate with how frequently the site gets cut,” says Joung.
He calls the process, named GUIDE-Seq, “comprehensive and sensitive,” and says it seems to be picking up mutation-causing edits of extremely rare frequency (“probably much lower” than 0.1 percent).
Some of Joung’s previous work has been licensed to Editas, but it’s unclear if GUIDE-Seq will follow suit. He and his colleagues will continue to refine it in at least two ways.
First, GUIDE-Seq needs to be tested in more “therapeutically relevant” cells, says Joung. The cells he used were old cancer cell lines that are great for research because they are practically immortal, but they don’t resemble anything you’d find in the real world—in part because of the very repair machinery they use to keep themselves going.
Second, GUIDE-Seq needs to be tested with a wider variety of guides and scissors. Right now, CRISPR/Cas9 tools come in mainly one “brand,” so to speak, derived from the bacterium Streptococcus pyogenes. But researchers including Joung are feverishly working to expand that toolkit, using enzymes from other bacteria (S. thermophilus, essential to yogurt and cheese production, is on Joung’s list) and building different kinds of guides that help the enzymes hone in on the right strands of DNA to cut.
Another new method for finding off-target cuts comes from a group of researchers in the Alt Laboratory at Boston Children’s Hospital. They measure instances, called chromosomal translocations, of major pieces of DNA being sheared off by the gene-editing scissors and moved to another part of the genome. That paper caught the attention of Matthew Porteus, a Stanford University researcher and doctor who treats children with hematologic cancers. “It’s as important as Keith (Joung)’s paper, or perhaps more,” says Porteus, who works with gene-editing technology and is helping the venture-backed Crispr Therapeutics, of London, develop a hematological product with CRISPR/Cas9 tools.
The paper suggests that gene-editing tools could create translocations between the target site and random sites in the genome, Porteus explains. “That needs exploration,” he says, because of a potential link between translocations and cancer. “What’s the functional consequence of a low frequency of translocations? Are they associated with cancer or of no consequence?” he asks. “We don’t have enough data.”
Porteus certainly wants better quality control systems. But he also cautions that the perfect should not be the enemy of the good. He counts himself among those who suspect off-target breaks from these new tools are something our bodies—our cells—already know how to deal with. “Cells are accumulating small insertions and deletions all the time,” says Porteus. “Our genome is not stable.” He’s not convinced that these new tools create more
