Adult female Aedes aegypti. Credit: James Gathany, CDC
There’s good reason why CRISPR-Cas9 gene editing is not allowed at the germline. While international commissions are working hard to make this a possibility, potential unknown effects further down the ancestry line raise concerns about the process.
The insect equivalent of this—gene drive transgene research—hasn’t been a cause of much concern for researchers working on genetic control of vector populations, especially disease-carrying mosquitoes. Scientists from Texas A&M, however, think the potential affects should be always be considered and have now devised a technology to make all genetic modifications in mosquitoes temporary—until a time when adequate testing ensures safety.
Zach Adelman, author of a new paper on the research and a professor at Texas A&M, says many of today’s insect genetic control strategies are based on highly invasive, self-propagating transgenes that can rapidly spread the trait into other populations of mosquitoes. Adelman’s method, however, allows proposed genetic changes to be tested on a temporary basis—without the risk of transmitting them to wild populations. The temporary genetic modifications self-delete over multiple generations of mosquitoes.
“There are lots of ecological questions we don’t know the answers to, and when you are testing technology, you don’t want to get into a situation where you have to tell a regulatory agency or the public that ‘if something bad happens, we’re just out of luck,’” Adelman said. “This mechanism is about how we get back to normal whether the experiment does or doesn’t come out the way we expect.”
In the study, published in PNAS, Keun Chae, a post-doctoral researcher in Adelman’s group, led the experiments in Aedes aegypti mosquitoes, known to transmit dengue virus, yellow fever virus, chikungunya virus and Zika virus. Taking advantage of a single-strand annealing (SSA)—a eukaryotic DNA repair mechanism—Chae engineered a duplicated genetic code region along with two genes for fluorescent proteins into the middle of a gene important for eye pigment.
The result was a white-eyed mosquito, and also red and green fluorescence in the eyes and body. When combined with a site-specific nuclease, which is essential for many aspects of DNA repair, they acted as a precise set of molecular scissors that could cut the transgene sequences. Over several generations, mosquitoes regained their normal eye pigment and lost the modified genes.
According to the paper, this suggests an alternative to how gene drive field trials could be conceptualized. Instead of a single trial format where uncertainty is high and removal of the gene drive transgene may not be possible, the successful proof-of-principle offers a two-step format where an initial trial is performed with the gene drive transgene bounded by the SSA-elimination mechanism.
“Whether the trial concludes as planned, is interrupted, or is stopped prematurely, the population reverts back to a non-transgenic state, eliminating the engineered transgenes and leaving just silent or neutral variants expected to mimic naturally occurring variants,” the researchers explain in their paper.
The mosquito genome is famously difficult to manipulate, and this breakthrough is the culmination of six years of experimental work. But it is still only the first step in a longer process—one that the research team believes will work on animals/insects other than mosquitoes.
“These are highly conserved genetic pathways, and there is every reason to believe this method could be applied to a diverse range of organisms,” said Kevin Myles, study author and a professor at Texas A&M.
The scientists say they are looking forward to expanding the application of their discovery in the context of highly active gene drive, hoping their method will be useful in pushing the boundaries of genetic research.