Kevin Mayer Senor Editor Genetic Engineering & Biotechnology News

Molecularly Editing Populations for Specific Benefits

A car pulls up alongside you. Its horn sounds, and its driver asks you to jump in. The destination? A genetically engineered heaven in which populations of organisms, even entire ecosystems, are altered to satisfy human needs and desires. Sounds great, except the driver admits he isn’t entirely sure of his route. He might take an unfortunate turn here and there, picking up and dropping off dangerous genetic hitchhikers, accidentally turning entire species into road kill, or getting lost in the pesticide-resistant weeds.

Yes, this could be a wild ride, but you might find it reassuring that the driver not only wants you in the passenger seat, he also wants you to be aware of the risks. He’s actually inviting you to be a backseat driver, someone who will tell him when to slow down, ask for directions, or head back to a missed exit.
The driver, you see, is behind the wheel of a powerful machine, so powerful that he feels he must warn you before he shifts it into high gear. The machine is a technology called gene drive.

In fact, gene drive is already accelerating because it now has the advantage of CRISPR/Cas9, a precise and efficient means of genomic editing. For example, on November 23, in the pages of the Proceedings of the National Academy of Sciences, scientists based at the University of California, Irvine reported that they combined gene drive and CRISPR/Cas9 and technologies to spread antimalarial genes through a population of mosquitoes.

A Peek under the Hood

Any genetic change that would propagate through a population faces a steep climb, particularly if the change confers little or no survival advantage. Even if such a change were to be passed from generation to generation, it would probably spread through a population too slowly to avoid being diluted through the workings of natural selection. And so there would seem to be little point in engineering genetic changes meant to be realized on the population scale.

But scientists can dream, can’t they? This particular dream goes back at least as far as the 1970s, the horse-and-buggy days of genetic manipulation, when scientists started considering transgenic updates to the sterile insect technique. In the original sterile insect technique, large numbers of insects were sterilized with radiation and released into populations of their wild counterparts. Irradiated, sterile insects mated with wild, fertile insects, forming unions that would fail to produce offspring.

This radiation-based technique had its successes. For example, it served to eliminate screwworm infestations of cattle. Yet it was self-limiting. If it could be put on a transgenic basis, scientists reasoned, it could become self-perpetuating. Deleterious genes could be made to spread through a population, rendering it harmless or even eliminating it.

Early transgenic technology was not up to the task, however, and the dream languished, except for a revival in 2003. That year, Austin Burt, Ph.D., an evolutionary geneticist at Imperial College London, found inspiration in a natural genetic mechanism, one that allows “selfish” genes to ascend daunting evolutionary gradients.

Dr. Burt was especially interested in site-specific selfish genes known as homing endonuclease genes (HEGs). Writing in the pages of the Royal Society of London B, Dr. Burt described how HEGs work: “They encode an enzyme that recognizes and cleaves a 20–30 bp sequence found on chromosomes not containing a copy of the HEG. The HEG itself is inserted in the middle of its own recognition sequence, and so chromosomes carrying the HEG are protected from being cut.”

“The broken HEG-negative chromosome will typically be repaired by the cell’s recombinational repair system, which uses the intact HEG-positive homologue as a template,” he continued. “After repair, both chromosomes will contain a copy of the HEG.”

This mechanism skews the odds of inheritance in sexually reproducing organisms. Ordinarily, a gene has a 50:50 chance of being passed on to individual offspring. But as part of an HEG construct, a gene can be passed on nearly 100% of the time. And so a selfish gene can drive itself into a population, even if it originates in just a few of the population’s members. Hence the name “gene drive.”

Dr. Burt pointed out that gene drives could be used to manipulate natural populations, but only if site-specific selfish genes such as HEGs could be engineered to target new host sequences. At the time, site-specific selfish genes could not be engineered with the necessary precision, and so field trials of population-altering technologies remained a distant prospect. Still, Dr. Burt anticipated that the “ease and rapidity with which these selfish genes can invade a population applies not only to planned releases, but also to unintentional releases of laboratory escapees.”

Dr. Burt’s paper attained landmark status in population genetics, and there is little wonder why. The paper not only described a technological approach to altering a population’s genetic makeup, he also forecast the rise of containment issues, as well as the need for criteria for deciding whether to eradicate or genetically engineer an entire species.

“Clearly, the technology described here is not to be used lightly,” he concluded. “Given the suffering caused by some species, neither is it obviously one to be ignored.”

That statement is even more apt today, now that gene drive is incorporating CRISPR/Cas9, a powerful gene-editing technology that can precisely insert, replace, and regulate genes. With CRISPR/Cas9, gene drive is like a vehicle revving its engine.

Pedal to the Metal

To fight malaria, some scientists are using CRISPR/Cas9-powered gene drive technology to do more than merely suppress mosquito populations. Instead, they are using it to bring parasite-resistance genes to mosquitoes and thereby render mosquito populations relatively harmless. For example, as mentioned above, this approach has been taken by scientists based at the University of California, Irvine. These scientists, led by Anthony James, Ph.D., published their findings in the Proceedings of the National Academy of Sciences (PNAS) in an article entitled “Highly efficient Cas9-mediated gene drive for population modification of the malaria vector mosquito Anopheles stephensi.”

“[Parasite-resistance] genes have been developed for the human malaria parasite Plasmodium falciparum,” wrote the article’s authors. “[We] provide evidence for a highly efficient gene-drive system that can spread these antimalarial genes into a target vector population.”

According to the scientists, their CRISPR/Cas9 system succeeded in a proof-of-concept experiment that was focused on the vector’s germ line. Their system was able to copy a genetic element from one chromosome to its homolog, the scientists asserted, “with more than 98% efficiency while maintaining the transcriptional activity of the genes being introgressed.”

The effector genes were observed to remain transcriptionally inducible upon blood feeding. However, to optimize the gene drive in wild mosquitoes, it will be necessary to overcome an unforeseen complication: a drive-dampening maternal effect on Cas9 expression. A press announcement issued by the PNAS noted that while the study looked promising, it would have to be supported by additional studies on the stability of effector genes in different mosquito strains. The PNAS also cited the need for research to validate gene drive performance in different environmental conditions.

Are We There Yet?

The implications of CRISPR/Cas9-powered gene drives were discussed at a workshop organized by the Woodrow Wilson Center and MIT in January 2014. Some of the implications were positive—combating malaria and other insect-borne diseases, controlling invasive species, and promoting sustainable agriculture. Some were disturbing. For example, gene drives meant to affect just one species might move to related species, potentially instigating a cascade of unintended effects. Or gene drives might weaken or eliminate a harmful species to create an ecological vacuum that could, perhaps, be filled by something just as bad, or even worse.

Later that year, these issues were addressed by a pair of papers. The first paper, published in eLife (“Concerning RNA-guided gene drives for the alteration of wild populations”), described the proposed technical methods of building gene drives in different species, defined their theoretical capabilities and limitations, outlined possible applications, and called for means of reversing gene drives. The second paper, featured in Science (“Regulating gene drives”), provided an initial assessment of potential environmental and security effects, an analysis of regulatory coverage, and recommendations to ensure responsible development and testing prior to use.

When these papers appeared, the Wyss Institute issued a press release that made it clear that the papers’ authors invited public scrutiny of gene drives. The release quoted the Wyss Institute’s Kevin Esvelt, Ph.D., as follows: “We all rely on healthy ecosystems and share a responsibility to keep them intact for future generations. Given the broad potential of gene drives to address ecological problems, we hope to initiate a transparent, inclusive and informed public discussion—well in advance of any testing—to collectively decide how we might use this technology for the betterment of humanity and the environment.”

Now, more than a year later, the public may be catching up. On November 10, the New York Times ran an op-ed entitled, “The Risks of Assisting Evolution.” This op-ed, to its credit, distinguished potential gene drive issues from other genetic-engineering controversies, such as the modification of human germline cells, or the release of mosquitoes incorporating genetic changes that can be passed on just once, not multiple times from generation to generation.

Earlier this year, when Chinese scientists used a CRISPR/Cas9 system to replace a gene implicated in β-thalassemia, they were widely criticized for working with human embryos, even though these embryos had been deemed nonviable, which meant that any modified genes could not have passed to successive generations. (Incidentally, this study did not deploy gene drive technology, which in any case lacks the power to alter human populations on anything close to a reasonable time scale, since human generations succeed each other so slowly.)

Similarly, when Oxitec proposed a field trial in which genetically modified mosquitoes would be released in Florida, outrage ensued, even though the effects of the release were self-limiting. (Although the field trial was to release male mosquitoes that were fertile, not sterile, the mosquitoes were to be genetically modified to carry a “dominant lethal” gene, which would lead to offspring with a high probability of dying.)

The Chinese research project and the proposed Oxitec release stimulated some fairly heated rhetoric against genetically modified organisms (GMOs). Whatever its merits, such rhetoric was loosed against studies of relatively modest scope. More ambitious studies, projects that would truly test the power of gene drive technology, might drive GMO critics around the bend.

To avoid such an eventuality—and prevent controversies that would generate more heat than light—gene drive experts are being proactive. They are frankly acknowledging gene drive’s inherent risks, and even anticipating safeguards that might be implemented. For example, on November 16, Wyss Institute researchers published an article in Nature Biotechnology on improved physical biocontainment barriers and the introduction of molecular confinement mechanisms.

A generation ago, such candor might have seemed unusual. Back then, critics of technology such as Jerry Mander complained that any new technology would typically arrive in a cloud of best-case scenarios, and that its drawbacks would become clear only after it was well entrenched. That’s not happening with gene drive. Still, cynics might say that early disclosures of gene drive’s risks could serve to inoculate gene drive's reputation against later revelations, should they grow increasingly dire, or encourage otherwise skeptical people to feel “embedded,” subtly enlisted in a common cause. So, gene drive could be a democratic road trip or a well-managed campaign. Your mileage may vary.

This article has been updated as of November 23, 2015 to reflect the publication of a gene drive study in the Proceedings of the National Academy of Sciences. The study, which is credited to Gantz et al., describes a prototype CRISPR/Cas9-based gene drive system for engineering malarial parasite resistance in mosquitoes.

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