Reversing insecticide resistance with a zero GMO endpoint

Background: Insects can be both our friends and enemies. On the one hand, they are essential components of diverse ecologies, playing key roles as pollinators of wild plants and agricultural crops. Additionally, they serve as important sources of protein for diverse organisms within the food web. On the other hand, some insects carry devastating human diseases including malaria and viruses such as those causing Dengue fever, Zika, Chikungunya, and hemorrhagic fevers. Insect pests also inflict grave agricultural damage either by devouring crops or by transmitting plant pathogens such as fungi or viruses. 

Control of insect disease vectors and agricultural pests relies primarily on heavy and repeated application of insecticides. This pervasive practice has two serious drawbacks. First, most insecticides indiscriminately kill both beneficial and harmful insects, leading to serious impacts on the environment. Second, the intensive use of these chemicals over the past several decades has selected for insecticide resistant (IR) mutants that have replaced the native populations of nearly all targeted insects. This escalating resistance, often to multiple pesticides, compels farmers and vector control agents to apply these poisonous compounds at yet great frequency and at ever increasing concentrations, creating severe collateral damage to the environment and human health. 

Developing new insecticides is also extremely expensive, costing over $250 million from discovery to production. No new classes of such compounds have been brought to market in over two decades. How can this vicious and costly IR cycle be broken? 

Previously, we engineered a multi-component genetic system referred to as an allelic-drive to reverse insecticide resistance due to mutations generated in the gene (vgsc) encoding the Voltage Gated Sodium Ion Channel (Kaduskar et al., 2022; Nat. Commun. 13, 291). Because the VGSC is the target of pyrethroid insecticides and DDT, a common IR mutation (L1014F) has arisen multiple time in the vgsc gene as a consequence of selection. Our CRISPR-based allelic-drive consisted of separate genetic elements encoding the Cas9 endonuclease and a guide RNA (gRNA) that selectively targeted the 1014F IR allele for DNA cleavage, while leaving the wild-type 1014L allele intact. This allelic-specific cleavage resulted in DNA repair of 1014F mutation using the 1014L allele as template. When all components of this allelic-drive system were introduced into an a freely mating population of fruit flies carrying the 1014F allele, it converted over 80% of the 1014F alleles back to 1014L over 8 generations. This system, although effective, left several genetic elements in the population, including a gene-drive cassette carrying two gRNAs to copy itself along with targeting the 1014F allele and a separate element expressing Cas9. Given public concerns regarding genetically modified organisms (GMOs), we wondered whether we might design a new transiently acting IR reversal system that would leave no GMO in the environment.

Current Study: We have now developed an efficient second generation allelic-drive approach to reverse insecticide resistance without creating any other perturbation to the environment. This novel precise intervention, referred to as a self-eliminating drive, relies on introducing insects carrying a short-lived genetic cassette that converts insecticide-resistant forms of mutated insect genes back to their original native form (Auradkar et al., 2024; Nat. Commun. 15, 9961). The evanescent genetic cassette is programmed to act transiently and then disappear from the population leaving only insects with the corrected normal version of the target gene.

Caption: Deployment of the Self-Elimination Drive leads to reversal of insecticide resistance at the vgsc locus.  Left panel: A population of insects carries the insecticide resistant (IR) vgsc^1014F allele, which confers knock-down resistance (kdr) to pyrethroid insecticides and DDT. Middle panel: Release of individuals carrying the Self-Elimination Drive converts the mutant IR vgsc^1014F population back to the wild-type insecticide sensitive (IS) state (vgsc^1014L) in approximately 6-months. Right panel: Low-level insecticide treatment leads to effective control of the IS population.

The self-eliminating drive system consists of only a single genetic cassette inserted into the genome at a location where it imposes a fitness cost on the host insect. In principle, this drive cassette could either reduce viability or fertility of individuals that carry the element. This system works as follows. When insects carrying the self-eliminating cassette are introduced into an insecticide resistant (IR) population, they mate randomly, transmitting the insecticide susceptible trait to nearly all of their offspring. The genetic element accomplishes this feat by harnessing the widely used CRISPR gene editing system to convert IR variants of the target locus back to its native or "wild-type" form. In these experiments, the target locus is the vgsc gene, which is widely required for nervous system function. The discriminating entity carried by the gene cassette is a guide RNA (gRNA) that binds to a DNA cutting protein called Cas9 (also carried on the cassette) and leads to selective cleavage of the non-preferred IR variant of the gene, but not of the wild-type form. The DNA break in the IR form of the gene is then repaired using information from an available wild-type copy of the gene thereby converting the previously IR variant back to its wild-type status. This process of target gene "editing" continues exponentially at each subsequent generation until all IR variants in the population have been reverted back to the native, insecticide susceptible form.

Because insects carrying the gene cassette are penalized with a severe fitness cost, the element is rapidly eliminated from the population lasting only as long as it takes to convert all IR forms of the target gene back to wild-type (in the laboratory experiments, this process took 8-10 generations - about six months for fast breeding insects such as flies or mosquitoes). In these experiments, the gene cassette was located on the X-chromosome and greatly reduced the mating success of males (which only have one X-chromosome). Females carrying the gene cassette, however, could mate readily with IR males, and thus were the agents responsible for reversing IR. But because males carrying the cassette did not have many progeny, the frequency of that cassette in the population dropped rapidly at each generation until it disappeared completely. 

An important feature of this ephemeral self-eliminating drive system is that it can be reused over and over again. This is important since this restorative genetic system can only be deployed during periods when the relevant insecticide is not in use (a different type of insecticide must be applied during the drive IR-reversal period). When the targeted insecticide used in subsequent years, it will be highly effective since nearly all insects will now be sensitive to it. Over time, however, those insects will become resistant once again to that insecticide as its application will select inevitably for such variants. Hence, the need to treat iteratively with a sustainable protocol is readily met with the self-eliminating drive system. Clean and simple, with no remaining genetic imprint on the environment - about as light a touch as could be achieved.

The work continues: Self-eliminating allelic-drive systems such as that described here, could be extended to reversing resistance to additional vgsc IR mutations or to those conferring resistance to other classes of insecticides (e.g., IR mutations in the gene encoding Acetylcholinesterase, which are selected for by use of organophosphates or carbamates). Similar systems could also be employed to rebalance the frequency of other naturally occurring allelic variants. For example, in anopheline mosquitos that transmit malaria, an allelic variant of the gene encoding the intestinally expressed FREP1 protein can confer resistance to parasite infection. Thus, an allelic-drive system that bias inheritance of a known parasite refractory FREP1 variant could potentially be employed in the global effort to eliminate malaria. These and other impactful applications of self-eliminating allelic-drives could usher in a new era of fine-touch genetic engineering to accomplish important health endpoints without leaving any genetic machinery in the environment.