ReMOT Control: Development of a flexible toolkit for the genetic manipulation of insects

The ability to change a gene or the way a gene is used by an organism is a cornerstone of molecular biology and underpins our fundamental understanding of many biological disciplines. Not only have these techniques illuminated our understanding of insects, but they also have profound implications for controlling insects in agriculture and public health. While altering the DNA of standard laboratory insect like the fruit fly (Drosophila) is routine, in most other insects it is a challenging and time-consuming process. In some cases it is simply not feasible due to the unique biology of the particular species. In many insects genes can also be silenced using a technique called RNAi, but this can be challenging in early life stages and efficiency can be low because RNA injected into the insect is not efficiently taken up by cells. Recently we developing a revolutionary approach to deliver cargo into developing eggs simply by injecting adult female insects. This has the potential to alleviate the challenges associated with gene editing and silencing in many insect species.

In many higher organisms, egg yolk proteins are synthesised in tissues of the mother and then transported to the developing eggs within the ovary. This relies on specific sequences in the yolk proteins that bind to receptors in the ovaries. We recognised that if we added this sequence to another molecule, we could hijack this process to deliver cargos into the egg as it develops. After identifying the specific sequence of the yolk protein that enables it to enter ovaries, we fused it to the protein Cas9 that can mutate specific genes within a genome. This allowed us to mutate a gene in mosquitoes that alters the colour of their eyes. The approach was highly efficient and we obtained mutants after injecting as few as ten females, which was a dramatic improvement on traditional approaches that rely on embryo microinjection. We named this technology Receptor-Mediated Ovary Transduction of Cargo (ReMOT Control). While the ReMOT Control technology is ground-breaking, and has the potential to democratize CRISPR-Cas9 editing approaches in high organisms, implementation is in its infancy. Here we will expand this technology into other insect species and broaden the molecular tools which can be delivered to the ovary by ReMOT Control.

We will exploit the molecular resources in Drosophila and the yellow fever mosquito (Aedes aegypti) to extend ReMOT Control to new forms of genetic manipulation. In our first aim, we will extend our existing techniques to insert specific DNA sequences into the fly and mosquito genomes, and test a variety of techniques to make this more efficient. We will then adapt a commonly used tool in fly genetics, the phiC31 system, to ReMOT Control to add genes into insect genomes. Using technology developed in the targeted drug delivery field, we will use ReMOT Control to deliver double stranded RNA and plasmids to the developing ovary for gene silencing and over-expression. Proof-of-principles experiments will be done by silencing of over-expressing fluorescent proteins and once optimized, we will confirm our ability to alter the expression of endogenous insect genes. Finally, we will identify proteins that are efficiently imported into the ovaries of other flies and mosquitoes, to allow the approaches to be extended to other medically or agriculturally important species. This project will enhance and expand ReMOT Control, changing the landscape of molecular entomology and provide a blueprint allowing the easy genetic manipulation of a wide variety of arthropods.

The CRISPR-Cas9 system has revolutionized genetics by allowing researchers to precisely modify the genome of essentially any organism. For most insects, current approaches rely on injecting the Cas9 endonuclease and guide RNA (or DNA encoding these) into embryos. In many species this is technically challenging and severely limits the scale and scope of genetic manipulations. We recently developed a revolutionary approach to efficiently mutate genes by delivering the Cas9 ribonucleoprotein (Cas9 and guide RNA) to oocytes by injecting adult females. This technology, which we termed ReMOT Control, relied on fusing Cas9 to a ligand from the Drosophila yolk protein that enters oocytes by receptor-mediated endocytosis. Here we will extend this technology to new forms of genetic manipulation and new species.

Our technology lends itself to delivering the molecules required for a wide range of genetic manipulations to developing embryos. Using fly and mosquito systems, we will first extend our existing Cas9 approach from simple mutagenesis to true genome editing involving the integration exogenous DNA. We will then adapt the technology both to alter gene expression by delivering RNAi and over-expression plasmids to embryos, and create transgenic insects using the phiC31 system. Finally, we will devise a straightforward strategy to identify ligands that can target cargos to the oocytes of a wide range of other Dipteran species, allowing the technique to be applied to other species of agricultural or medical importance. Together, this work will greatly enhance the molecular tool kit available to researchers working on a wide range of insect species.

Insects are of critical importance to life on earth. From a human perspective some species vector disease or are agricultural pests, while others like pollinators are beneficial. Regardless of their relationship with humans, tremendous insights into the biology of insects has been achieved with reverse genetic approaches, and in turn these insights have led to economic and public health advances. However, most of this work has been achieved in only a limited number of commonly studied species and many insects are recalcitrant to genetic manipulation. Transferring these technologies to other insect species would be tremendously significant and enhance our capacity to understand, utilize and control insects. We have developed a revolutionary new system to target cargo to the insect ovary which overcomes many of the current hurdles for editing the genomes or altering transcription of many insect species. Our work will build the foundation of new approaches to edit and manipulate the genetics of model and non-model insects alike, thus greatly expanding tools available to a range of scientific fields studying a broad spectrum of insect species. As such, our findings will stimulate research in these areas underpinning human health and food security.