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

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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.