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

点击翻译按钮获取中文摘要

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.