A technique for editing genes while they reside in intact chromosomes has been a real breakthrough. Literally. In 2013, Science magazine named it the runner-up for breakthrough-of-the-year, and its developers won the 2015 Breakthrough Prize.

The system being honored is called CRISPR/Cas9, and it evolved as a way for bacteria to destroy viruses using RNA that matched the virus' DNA sequence. But it's turned out to be remarkably flexible, and the technique can be retargeted to any gene simply by modifying the RNA. Researchers are still figuring out new uses for the system, which means there are papers coming out nearly every week, many of them difficult to distinguish.

That may be precisely why the significance of a paper published last week wasn't immediately obvious. In it, the authors described a way of ensuring that if one copy of a gene was modified by CRISPR/Cas9, the second copy would be—useful, but not revolutionary. What may have been missed was that this process doesn't stop once those two copies are modified. Instead, it happens in the next generation as well, and then the generation after that. In fact, the modified genes could spread throughout an entire species in a chain reaction, a fact that has raised ethical and safety concerns about the work.

New developments

The CRISPR/Cas9 system is remarkably simple. It relies on RNA molecules that have a specific format and are able to base pair with a site in the genome. Cas9 then cuts the DNA at the site where this base pairing occurs, creating a break in the chromosome. Cells have systems that attempt to repair these breaks, and these systems attempt to identify similar-looking sequences to use as a template for repair. So if you provide the cells with some similar DNA, it will end up being placed at the site that the RNA first targeted.

This makes it easy to modify the genome. By providing slightly different DNA to be used in the repair process, you can substitute altered bases, short deletions, or even entire additional genes, any of which can take their place within the chromosome. In short, CRISPR/Cas9 lets you put any DNA you want anywhere in a genome.

It's possible to use this to eliminate genes you're interested in, so you can study animals that lack that gene. You simply target the gene with an RNA, and then provide DNA with a deletion of a key part of the gene. The repair system will use the deletion as part of its template and copy it into place on the chromosome. It's also possible to mutate a gene by replacing key parts of it with something else. For example, you could swap in a copy of the Green Fluorescent protein and ensure that all of the resulting mutants glow green.

But you still have to breed these mutations the old-fashioned way: you need to get two organisms that have a copy of the mutant gene, then breed them together. Mendel then tells us that one-quarter of the offspring will have mutant copies in both of their chromosomes.

The authors of the new paper found that frustratingly slow. Working in flies, they designed a system where CRISPR/Cas9 would do all the work for them. Their DNA repair template was a bit more complicated than a simple deletion. Instead, it contained the genes needed to get the CRISPR/Cas9 system to work, along with a guide RNA that targeted a specific fly gene (in this case, yellow). They surrounded all these genes with DNA from the yellow gene itself.

Once injected in the fly, the normal yellow gene was disrupted by the genes for the CRISPR/Cas9 system. Once that happened on one chromosome, the system could easily perform the same modification on the other chromosome, making the animal a homozygous yellow mutant.

But the key thing is what happens in the next generation. In these animals, a normal copy of the yellow gene comes in from the next parent. But the CRISPR/Cas9 cassette immediately converts that, too, resulting in offspring that are all yellow. Well, not all; but the authors found that the construct was 97 percent effective at converting the next generation. In fact, there's nothing to stop this system from invading an entire population, continuing to convert generation after generation until everything carries the modification.

It's a bit like the futurists' fears of a self-perpetuating "grey goo," just played out with yellow-colored flies. (For those of you with a biology background, this will also sound a lot like an engineered homing intron.)

Precautions

Fortunately, the researchers were conscious of the issues: "we are also keenly aware of the substantial risks associated with this highly invasive method since the failure to take stringent precautions could lead to the unintentional release of [modified] organisms into the environment." The flies were bred behind three layers of containment in a locked facility. The containers were put straight into the freezer to kill the flies if they were no longer needed. Any manipulations of the flies were performed while they were anesthetized in a Biosafety Level 2 facility.

Still, there are further precautions that could be taken. The report cites a draft manuscript, hosted on the bioRxiv, that describes a similar system in yeast. In this case, however, only the targeting RNA is inserted into the targeted gene—the rest of the CRISPR/Cas9 system has to be provided separately for anything to happen. One of the authors of this manuscript, the synthetic biologist George Church, told a Science reporter that he felt the fly work should never have been published because the technology was too dangerous.

Why do the work at all if it's so risky? Because, properly controlled, there could be some amazing benefits. Imagine using it to quickly breed traits from non-agricultural plants (drought or pest resistance, for example) into important food crops. Or converting the entire population of a dangerous pathogen into one lacking virulence genes. Or releasing a few mosquitos, allowing them to breed, and creating a population that's incapable of supporting malarial parasite growth. All of these are very real possibilities enabled by the technology.

But there's also a very real risk of a giant, uncontrolled experiment if any of these DNA constructs made it into a wild population. And the developments come at a time where several researchers (including Church) have suggested it's time to lay out some formal guidelines for future research in this area, both for synthetic biology and for human genome modifications. The authors of the fly paper cite two of these editorials, while Science and Nature have run editorials urging that we avoid editing the human germline.

The authors of the fly paper suggest looking to the Asilomar agreement, which was forged by leading biologists who were leaders in the development of recombinant DNA. That created a voluntary yet successful moratorium on the work until safety issues could be examined. We may be forced to see whether this sort of voluntary agreement would hold in an era of intense competition. Similar concerns were voiced about work involving flu viruses, but research continued until the federal government announced a halt to funding for this research.

Science, 2015. DOI: 10.1126/science.aaa5945 (About DOIs).