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

改变基因或基因被生物体使用的方式的能力是分子生物学的基石，也是我们对许多生物学科的基本理解的基础。这些技术不仅启发了我们对昆虫的理解，而且对农业和公共卫生中的昆虫控制也有深远的影响。虽然改变果蝇（Drosophila）等标准实验室昆虫的DNA是常规的，但在大多数其他昆虫中，这是一个具有挑战性且耗时的过程。在某些情况下，由于特定物种的独特生物学特性，这根本不可行。在许多昆虫中，基因也可以使用称为RNAi的技术沉默，但这在生命早期阶段可能具有挑战性，并且效率可能很低，因为注入昆虫的RNA不能有效地被细胞吸收。最近，我们开发了一种革命性的方法，通过注射成年雌性昆虫将货物运送到发育中的卵子中。这有可能减轻许多昆虫物种中与基因编辑和沉默相关的挑战。在许多高等生物中，卵黄蛋白在母体组织中合成，然后运输到卵巢内发育的卵子。这依赖于蛋黄蛋白中与卵巢受体结合的特定序列。我们认识到，如果我们将这个序列添加到另一个分子中，我们可以劫持这个过程，在鸡蛋发育的过程中将货物运送到鸡蛋中。在确定了使其能够进入卵巢的卵黄蛋白的特定序列后，我们将其与可以使基因组内的特定基因突变的蛋白Cas9融合。这使我们能够突变蚊子的一种基因，从而改变它们眼睛的颜色。这种方法非常有效，我们在注射了10只雌性后就获得了突变体，这对依赖胚胎显微注射的传统方法来说是一个巨大的改进。我们将这项技术命名为受体介导的卵巢货物转导（ReMOT控制）。虽然ReMOT Control技术是突破性的，并且有可能使高等生物中的CRISPR-Cas9编辑方法民主化，但实施仍处于起步阶段。在这里，我们将把这项技术扩展到其他昆虫物种，并扩大可以通过ReMOT Control传递到卵巢的分子工具。我们将利用果蝇和黄热病蚊子（埃及伊蚊）的分子资源，将ReMOT控制扩展到新形式的遗传操作。在我们的第一个目标中，我们将扩展现有的技术，将特定的DNA序列插入苍蝇和蚊子的基因组中，并测试各种技术，使其更有效。然后，我们将调整苍蝇遗传学中常用的工具phiC 31系统，以ReMOT Control将基因添加到昆虫基因组中。利用靶向药物递送领域开发的技术，我们将使用ReMOT Control将双链RNA和质粒递送到发育中的卵巢中，用于基因沉默和过表达。将通过沉默过表达的荧光蛋白进行原理验证实验，一旦优化，我们将证实我们改变内源性昆虫基因表达的能力。最后，我们将确定有效输入其他苍蝇和蚊子卵巢的蛋白质，以便将这些方法扩展到其他医学或农业重要物种。该项目将加强和扩大ReMOT控制，改变分子昆虫学的景观，并提供一个蓝图，允许各种各样的节肢动物的简单遗传操作。

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.