Resistance to CRISPR gene drives may arise easily

A genetic-engineering tool designed to spread through a population like wildfire — eradicating disease and even whole invasive species — might be more easily thwarted than thought.

Resistance to the tools, called CRISPR gene drives, arose at high rates in experiments with Drosophila melanogaster fruit flies, researchers at Cornell University report July 20 in PLOS Genetics. Rates of resistance varied among strains of fruit flies collected around the world, from a low of about 4 percent in embryos from an Ithaca, N.Y., strain to a high of about 56 percent in Tasmanian fruit fly embryos.
“At these rates, the constructs would not start spreading in the population,” says coauthor Philipp Messer, a population geneticist. “It might require quite a bit more work to get a gene drive that works at all.”

Gene drives are basically genetic copy-and-paste machines. These self-perpetuating machines are inherited by more than 50 percent of offspring of an individual carrying a gene drive. Working perfectly, they could transmit to 100 percent of offspring.

In its simplest form, a CRISPR gene drive consists of a piece of DNA that encodes both an enzyme called Cas9, which acts as molecular scissors, and a guide RNA that tells the Cas9 enzyme where to cut. That cutting may disrupt important genes. Researchers are experimenting with this as a way to sterilize malaria-carrying mosquitoes (SN Online: 12/7/15).

Some gene drives also carry a genetic payload. For instance, another approach to fighting malaria is to develop drives that carry genes to “vaccinate” mosquitoes against the disease (SN: 12/26/15, p. 6). Other drives might carry genes that make fluorescent proteins to indicate the gene drive’s presence; Messer and colleagues used such markers to follow two gene drives in fruit flies bred in the lab.
When an organism carrying the tool mates with one that doesn’t, gene drives go to work. Inside the fertilized egg, guide RNAs shepherd Cas9 produced by the engineered mate to a spot where it cuts the other mate’s chromosome.

If everything works correctly, cells repair that break by copying the gene drive onto the cut chromosome. But the slice can also be fixed by gluing the cut ends back together. That regluing sometimes leads to mistakes that destroy Cas9’s cutting site, creating a chromosome that is resistant to the gene drive’s insertion.

In the fruit fly experiments, some mistakes created resistance during or before fertilization. Others took place in early embryos because cells produced Cas9 for too long, allowing the enzyme to chop chromosomes again and again, Messer and colleagues discovered. That was especially a problem when females produced Cas9, they found.

Some uses of gene drives, such as those that would sterilize or kill mosquitoes, can’t tolerate any amount of resistance no matter when it arises, Messer says. Because those types of gene drives damage the organism’s fertility or viability, mosquitoes carrying resistance would have an advantage and quickly outcompete insects vulnerable to the drives.

In a separate study posted June 14 at BioRxiv.org, Messer and colleagues tested several approaches to overcoming gene drive resistance. They found that using multiple guide RNAs and turning on Cas9 only in males could reduce resistance rates.

“This is a very important and elegant set of experiments,” says MIT evolutionary engineer Kevin Esvelt.

But the conclusions aren’t news to most gene drive researchers.

“We’re aware of all these problems, and the essence of how to deal with them hasn’t been changed by these studies,” says geneticist Ethan Bier of the University of California, San Diego. Bier and lab colleague Valentino Gantz created the first gene drive in fruit flies in 2015, and have worked with other researchers to develop gene drives that would prevent mosquitoes from carrying malaria (SN: 12/12/15, p. 16).

Messer’s group is, however, the first to experimentally confirm predictions about resistance and how to avoid it, Esvelt says. “They show what’s been apparent to some people in the field for a very long time.”

Some people might think that high rates of resistance mean that gene drives are safe to release because they won’t spread easily in the wild. But that notion is misguided, says Bier. Even if a gene drive is able to affect only a small percentage of a local pest population, it could still spread around the world, Esvelt adds. “It could still screw us all over in the current form.”

Researchers should continue to conduct gene drive experiments under tight containment, he and Bier caution.