Gene editing with the CRISPR-Cas9 system was already all the buzz before it alighted on the mosquito genome. But now the system has a taste for blood, so to speak. It has shown promise as a means of creating transgenic mosquito strains, enabling novel mosquito-control or disease-prevention strategies. And now CRISPR-Cas9, streamlined for use with mosquitoes, is prepared to elude the natural repair mechanisms that mosquitoes use to swat it down.
The streamlining has been carried out by scientists at Virginia Tech’s Fralin Life Science Institute. These scientists, reporting in Proceedings of the National Academy of Sciences, say that they have developed and validated a two-step process for performing high-efficiency site-specific insertion of genetic material into the mosquito genome.
The first step: Evaluate candidate site-specific nucleases in a rapid format, a transient embryo assay. The candidate nucleases of the CRISPR-Cas9 system rely on the integration of effective guide RNAs, the identification of which has tended to be a time-consuming chore.
The second step: Germ-line based editing where the choice of DNA repair response is constrained. Specifically, the Virginia Tech scientists found a way to silence an important mosquito DNA repair mechanism, ensuring that changes introduced by means of gene editing are more likely to become a permanent part of the mosquito genome, and thus increasing opportunities to figure out which genetic alterations cause desirable changes in mosquito biology.
The details appeared March 16 in an article entitled, “Silencing of end-joining repair for efficient site-specific gene insertion after TALEN/CRISPR mutagenesis in Aedes aegypti.”
“By first evaluating candidate guide RNAs using a transient embryo assay, we were able to rapidly identify highly effective guide RNAs,” wrote the authors. “Focusing germ line-based experiments only on this cohort resulted in consistently high editing rates of 24–90%.”
“RNAi-based suppression of Ku70 [an essential component of the mosquito’s end-joining repair mechanism] concurrent with embryonic microinjection of site-specific nucleases yielded consistent gene insertion frequencies of 2–3%,” the authors continued, “similar to traditional transposon- or ΦC31-based integration methods but without the requirement for an initial docking step.”
The authors concluded that their gene-editing model should significantly accelerate both basic and applied research concerning disease vector mosquitoes.
“We've cut the human capital it takes to evaluate genes in disease-carrying mosquitoes by a factor of 10,” said Zach N. Adelman, Ph.D., an associate professor of entomology in the College of Agriculture and Life Sciences and a member of the Fralin Life Science Institute. “Not a lot of research groups have the resources to spend four months working with up to 5,000 mosquito embryos to investigate a gene that may ultimately have no bearing on their work. Now they can potentially do the same investigation in a week.”
“The mosquito is incredibly important as far as transmission of disease,” said Kevin M. Myles, Ph.D., also an associate professor of entomology in the College of Agriculture and Life Sciences and a member of the Fralin Life Science Institute. “With this model, any scientist asking a question about a mosquito phenotype can now find its genetic cause. That's important for basic research into mosquito biology and applied research to control disease-vector mosquitoes.”
Mosquitoes transmit pathogens that cause malaria, dengue fever, and other high-impact diseases. In 2013, Malaria killed an estimated 584,000 people, most of them young children, according to the World Health Organization. Bill Gates, the co-founder of Microsoft and a philanthropist who supports social and health causes, has called the mosquito the world's deadliest animal.
“I am excited by the paper,” said Laura C. Harrington, Ph.D., a professor and the chairwoman of entomology at Cornell University, who was not involved in the research. “We are desperately in need of faster and more efficient ways to transform mosquitoes. This single hurdle is holding the entire field back from making the discoveries that will lead to novel and effective approaches to mosquito and, consequently, disease control.”
A lesser hurdle may be the need to clear field studies with people who distrust genetically modified organisms. For example, proposed field trials in Florida aroused opposition from local residents who doubted the safety of controlled releases of genetically modified mosquitoes.