IT IS life’s lottery, blessing some and cursing others in equal number: the chance of a sexually reproducing organism’s offspring inheriting a particular version of a gene from a particular parent is 50%. Usually. But there are exceptions. Gene drives are stretches of DNA that change those odds to favour one parent’s version of a gene over the other’s. That version will thus tend to spread through a population. If the odds are stacked sufficiently in its favour it can do so fast and, within a few generations, become the only version of the gene in question that remains in circulation.

Researchers realised, soon after the discovery of gene drives half a century ago, that they might be forged into tools for eradicating diseases and pests. For example, a drive promulgating a genetic variant that made mosquitoes unable to host the parasite that causes malaria could be used to help eliminate the disease. If the propagating variant made female mosquitoes sterile, it might provide a means to eliminate the troublesome insects themselves.

Engineering gene drives to do humanity’s bidding in this way proved, however, devilishly difficult. The idea therefore languished until 2015, when Valentino Gantz and Ethan Bier of the University of California, San Diego, used CRISPR-Cas9, a recently discovered gene-editing tool, to make a gene drive that could be inserted anywhere in a target genome that they chose.

Those findings sparked concerns about the effects gene-drive-carrying organisms could have if they were ever to be released into the world. For example, a gene drive that somehow hopped from a target species into the genomes of other animals might wipe them out before anything could be done about it. A study published in PLOS Genetics, by Philipp Messer of Cornell University and his colleagues suggests, however, that those who would deploy gene drives against scourges such as malaria face a more immediate hurdle: such drives simply may not work. Just as insects and pathogens evolve resistance to new pesticides and antibiotics, so gene drives, too, may provoke resistance—and may do so far faster than many suspected.

Life finds a way

In nature, elements of the CRISPR-Cas9 system help bacteria to ward off viral infections. Some viruses replicate themselves by inserting their DNA into the genomes of their hosts. This hijacks a cell’s protein-making machinery, causing it to turn out components for new viruses. CRISPR-Cas9 selectively excises such foreign DNA, eliminating the invaders.

The excision mechanism consists of two molecules: an enzyme (Cas9) derived from a bacterium called Streptococcus pyogenes, and a piece of RNA, a chemical akin to DNA that is made up of similar genetic letters. This “guide” RNA will stick only to a section of DNA with a letter-sequence complementary to its own. The enzyme is bound to the guide RNA. The guide RNA is bound to the DNA. Hence the enzyme recognises the DNA it has to cut, and snips it at the correct location. By inserting the gene for Cas9 into an organism, together with genetic material that instructs a cell to produce the correct guide RNA, biologists can therefore use CRISPR-Cas9 to sever a genome at a specific point.

They can then insert at that point whatever payload of other genes they might like—to modify mosquitoes so they cannot transmit diseases, say—knowing that the cell’s DNA-repair mechanisms will subsequently kick in to repair the incision around the newly inserted genes.

Handily, the system works in any organism, not just the bacteria in which Cas9 is found naturally. To use it as a gene drive, all that is required is to include in the payload the genes for the CRISPR-Cas9 system itself, and to ensure that some copies get inserted into an organism’s germ cells—those that develop into sperm or eggs. This will mean that the gene drive is passed on to all of that organism’s offspring.

CRISPR-Cas9 gene drives have, though, a significant drawback. When the drive cuts the genome but fails, for some reason, to insert itself into the incision, the cell instead inserts new genetic letters to replace those cut away by the enzyme before it rejoins the severed DNA strands. This process often changes the letter sequence at the site. That means the guide RNA can no longer recognise it. And that, in turn, means the organism (and its progeny) are now resistant to the drive.

Dr Messer wanted to understand this process. To do so, he and his colleagues observed the effects of introducing a CRISPR-Cas9 gene drive they had developed into fruitflies—insects commonly used in genetic studies.

Their drive’s payload was a gene that encodes red fluorescent protein, a substance normally found in a species of sea anemone. By further genetic tweaking, the researchers arranged for this protein to be expressed, in particular, in the insects’ eyes. They thus knew that flies with fluorescent eyes carried their gene drive. Also, the guide RNA they selected meant the drive inserted this payload into the middle of a gene named “yellow”, thus disrupting that gene’s action. Flies which inherit a defective version of “yellow” have yellow bodies, rather than black ones.

After experiments with thousands of flies, Dr Messer and his team found that the gene drive successfully inserted itself into the insects’ DNA about half the time, producing flies with fluorescent eyes and yellow bodies. The other half of the insects nearly all had yellow bodies, but did not have fluorescent eyes. That indicated CRISPR-Cas9 had cut the DNA in the right place, thus disrupting the function of “yellow”, but had failed to insert itself into the incision. Sequencing the genomes of these flies confirmed that in virtually all cases the consequence was a mutation that rendered the flies (and would have rendered any offspring) resistant to the gene drive.

Red signal

The team then repeated their experiments with fruitfly lines from five continents. They found that the proportion of flies becoming resistant to the drive varied from about 60% down to 4%. Differences in resistance to the drive were not caused by any initial differences in the target sequence. That did not vary between the five lines. They must therefore have stemmed from other (as yet unknown) genetic differences between the flies. This is a worry because it suggests that even if a drive works well in laboratory animals, it may fail in the wild when it encounters populations with higher resistance. Getting to the bottom of what is causing some lines to be more resistant than others will be an important step towards the development of gene drives that can spread traits through a species, Dr Messer reckons.

Two further modifications of CRISPR-Cas9 gene drives may help. The first is to equip them with several guide RNAs, allowing Cas9 to cut chromosomes at more than one place. An organism would have to develop resistant DNA sequences at all of these to become fully immune to the drive. Dr Messer and his colleagues have made such a drive, containing two guide RNAs, and have found that it did indeed lower the proportion of flies that developed resistance—though estimates made with computer models suggest this is still not enough for the drive to reach more than about half the population.

The second approach is to put the drive into the middle of a gene that, unlike “yellow”, an organism needs to survive. This might be expected to disrupt the gene and kill the organism. But if the inserted DNA has, at one end, a replica of the part of the disrupted gene that has been displaced by the insertion, this can meld seamlessly with its counterpart in the animal, preserving the gene’s function and Cas9’s ability to recognise it. If the join is not seamless, though, the gene will fail and the animal will die. Mutant genes resistant to the drive will thus be unable to spread.

At least two groups, one based at Imperial College, in London, and the other at Harvard University and the Massachusetts Institute of Technology, are working on drives aimed at essential genes in mosquitoes and which use multiple guide RNAs. If they succeed, they will breathe new life into the field. As Dr Messer observes, “it is difficult to cheat evolution.” Whether it is impossible to do so remains to be seen.