IT IS risky to predict who and what will win a Nobel prize. But some discoveries are so big that their receipt of science’s glitziest gong seems only a matter of time. One such is CRISPR-Cas9, a powerful gene-editing technique that is making the fraught and fiddly business of altering the genetic material of living organisms much easier.

Biologists have taken to CRISPR-Cas9 with gusto, first with animal experiments and now with tests on humans. In March researchers in China made history when they reported its first successful application to a disease-causing genetic mutation in human embryos. But their results were mixed. Although they achieved 100% success in correcting the faulty gene behind a type of anaemia called favism, they tested the technique in only two affected embryos. Of four others, carrying a mutation that causes thalassaemia, another anaemia, only one was successfully edited.

Now, in a study just published in Nature, a group of researchers from America, China and South Korea have pulled off a similar trick, with striking consistency, among many more embryos, while avoiding or minimising several of the pitfalls of previous experiments. Their work suggests that, with a bit of tweaking and plenty of elbow grease, CRISPR-Cas9 stands a good chance of graduating, sooner or later, from the laboratory to the clinic.

The researchers involved, Hong Ma of Oregon Health & Science University and her colleagues, obtained sperm donated by a man who carries a mutated version of a gene called MYBPC3 that causes hypertrophic cardiomyopathy (HCM), a condition in which the walls of the heart grow too thick. As with the genes that cause thalassaemia and favism, inheriting even a single copy of the malformed version of this gene is enough to cause HCM.

These sperm, half of which would have been carrying the mutated version of MYBPC3, were then used to fertilise eggs containing a normal copy of the gene. The resulting embryos thus had a 50:50 chance of containing a defective copy. In the absence of editing, and had they been allowed to develop, those with a faulty version would have grown into adults likely to suffer from the disease.

Swords to ploughshares

CRISPR-Cas9 editing has been developed from a bacterial defence system that shreds the DNA of invading viruses. CRISPR stands for “clustered regularly interspaced short palindromic repeats”. These are short strings of RNA, a molecule similar to DNA, each designed to fix onto a particular segment of a virus’s DNA. Cas9 is an enzyme which, guided by CRISPRs, cuts the DNA at the specified point.

Modifying this arrangement for the purposes of genetic engineering is simple, at least in theory. Since DNA and RNA work in essentially the same ways in all living organisms, designing appropriately customised CRISPR guide molecules can induce Cas9 to cut any cell’s DNA wherever the designers choose, eliminating undesirable sequences of genetic “letters”. Since cells will then try to repair this sort of damage, genetic engineers can, by providing corrected versions of the DNA that has been deleted for use as templates which a cell can copy, encourage the repair mechanism to fix the problem in the way they had intended.

The hope was that, by being given such templates, embryos could be purged of nascent genetic disease. That hope appeared fulfilled, at least in part. By the end of the experiment, 72% of the embryos were free of mutant versions of MYBPC3, an improvement on the 50% that would have escaped HCM had no editing taken place.

In achieving this, Dr Ma and her colleagues overcame two problems often encountered by practitioners of CRISPR-Cas9 editing. One is that the guidance system may go awry, with the CRISPR molecules leading the enzyme to parts of the genome that are similar, but not quite identical, to the intended target. Happily, they found no evidence of such off-target editing.

A second problem is that, even if the edits happen in the right places, they might not reach every cell. Many previous experiments, including some on embryos, have led to mosaicism, a condition in which the result of the editing process is an individual composed of a mixture of modified and unmodified cells. If the aim of an edit is to fix a genetic disease, such mosaicism risks nullifying the effect.

Dr Ma and her colleagues conjectured that inserting the CRISPR-Cas9 molecules into the egg simultaneously with the sperm might help. That way the process is given as much time as possible to complete its work before the fertilised egg undergoes its first round of cell division. Sure enough, after three days (by which time the original fertilised egg had divided several times), all but one of the 42 embryos in which the technique had worked showed the same modifications in every one of its cells.

So far, so good. But a third problem that has bedevilled experiments with CRISPR-Cas9 concerns the quality of the repair. There are at least two ways for cells to repair DNA damage. One of them simply stitches the severed strands of DNA back together, deleting or adding genetic letters at random as it does so. Because it introduces mutations of its own, this process is not suitable for correcting DNA defects for medical purposes (though it might, for instance, be used to modify crops). Fortunately, the other mechanism patches the break with guidance from a template, and thus without introducing any additional mistakes. But cells seem to prefer the slapdash approach. In previous CRISPR-Cas9 research, the more precise method was involved only 2% to 25% of the time.

Running repairs

The researchers’ cells were, however, much more diligent. That is, perhaps, to be expected. Any DNA damage to a fertilised egg which is not fixed properly will affect the entire organism, so embryos have an evolutionary incentive to get things right. But there was a surprise. Contrary to expectations, it was rarely the injected template that the cells used as a reference for repair. Of the 42 modified embryos, only one did so. The rest repaired the faulty gene by referring to the non-mutated copy they had inherited from their mothers. That contrasted with the results of control experiments the researchers carried out in parallel on human stem cells, in which the repair template they supplied was used much more frequently. This, they say, suggests a hitherto-unknown DNA repair mechanism may be at work in embryos.

If true, that is both good news and bad. It is good because it suggests embryos will often perform high-quality repairs without any extra prompting. It is bad because that repair will only be useful if the second copy of the gene is itself not harmful. Embryos that inherit two damaged copies of a gene, one from each parent, would simply replace one defective copy with another, to no overall benefit.

Jin-Soo Kim, of the Institute for Basic Science, in South Korea, who is another of the paper’s authors, thinks that, with a bit more research, genetic engineers may be able to get around that problem. He points out that mouse embryos seem to have no difficulty using external genetic templates. It may be that there are biochemical cues which control how a cell effects DNA repair, and that these can be manipulated. On the other hand, the difference may reflect an unbridgeable evolutionary divergence between mice and humans—species whose most recent common ancestor lived more than 60m years ago.

But that is a question for another paper. Over the coming months Dr Ma and her colleagues plan to replicate and extend their work using other mutations and other donors. One goal is to improve the process’s efficiency still further. Shoukhrat Mitalipov, a colleague of Dr Ma’s in Oregon, and yet another of the paper’s authors, thinks the technique’s rate of effectiveness can be boosted to at least 90%. The eventual objective, still a long way off, is full-blown clinical trials, in which modified embryos, purged of disease-causing genes, are reimplanted into their mothers and carried to term. If and when this is done successfully, human genetic engineering will truly have come of age.