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

ReMOT Control：开发用于昆虫基因操作的灵活工具包

基本信息

批准号：
BB/T001240/1
负责人：
Grant Leslie Hughes
金额：
$ 73.35万
依托单位：
Liverpool School of Tropical Medicine
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2020
资助国家：
英国
起止时间：
2020 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=BB%2FT001240%2F1
关键词：
ReMOT Control Development flexible toolkit

项目摘要

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.

改变基因或有机体使用基因的方式的能力是分子生物学的基石，并巩固了我们对许多生物学学科的基本理解。这些技术不仅启发了我们对昆虫的理解，而且对农业中的昆虫控制和公共卫生也有深远的影响。虽然改变标准实验室昆虫(如果蝇)的DNA是例行公事，但对大多数其他昆虫来说，这是一个具有挑战性和耗时的过程。在某些情况下，由于特定物种独特的生物学特性，这是根本不可行的。在许多昆虫中，也可以使用一种名为RNAi的技术来沉默基因，但这在生命的早期阶段可能会具有挑战性，效率可能会很低，因为注入昆虫体内的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，只需注射成年雌性昆虫就可以将货物运送到发育中的卵子中。这有可能缓解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，卵黄蛋白在母亲的组织中合成，然后运输到卵巢内的发育中的卵子。这依赖于卵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，在卵子发育时将货物运送到卵子中。在确定了使其能够进入卵巢的卵黄蛋白的特定序列后，我们将其与可以突变基因组中特定基因的蛋白Cas9融合。这使得我们能够突变蚊子的一种基因，这种基因可以改变蚊子眼睛的颜色。这种方法效率很高，我们只注射了10只雌性就获得了突变，这与依赖胚胎显微注射的传统方法相比是一个巨大的进步。我们将这项技术命名为受体介导的卵巢货物转导(Remot Control)。虽然Remot Control技术是开创性的，并有可能使CRISPR-Cas9编辑方法在高等生物体中大众化，但实施还处于初级阶段。在这里，我们将把这项技术扩展到其他昆虫物种，并扩大可以通过远程控制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，将远程控制扩展到新的遗传操作形式。在我们的第一个目标中，我们将扩展我们现有的技术，将特定的DNA序列插入苍蝇和蚊子的基因组中，并测试各种技术以使这一过程更加有效。然后，我们将采用苍蝇遗传学中常用的工具phiC31系统来远程控制，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和质粒运送到发育中的卵巢，以实现基因沉默和过度表达。原理验证实验将通过沉默过度表达的荧光蛋白来进行，一旦优化，我们将确认我们改变内源昆虫基因表达的能力。最后，我们将确定可以有效地输入到其他苍蝇和蚊子卵巢中的蛋白质，以便将这种方法扩展到其他具有医学或农业重要性的物种。该项目将增强和扩展远程控制，改变分子昆虫学的格局，并提供一张蓝图，允许对各种节肢动物进行轻松的基因操作。

项目成果

期刊论文数量（10）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Variable microbiomes between mosquito lines are maintained across different environments.

DOI：
10.1371/journal.pntd.0011306
发表时间：
2023-09
期刊：
PLoS neglected tropical diseases
影响因子：
3.8
作者：
通讯作者：

The microbiome and mosquito vectorial capacity: rich potential for discovery and translation.

DOI：
10.1186/s40168-021-01073-2
发表时间：
2021-05-18
期刊：
Microbiome
影响因子：
15.5
作者：
Cansado-Utrilla C;Zhao SY;McCall PJ;Coon KL;Hughes GL
通讯作者：
Hughes GL

Additional file 4 of Interspecies microbiome transplantation recapitulates microbial acquisition in mosquitoes

种间微生物组移植的附加文件 4 概括了蚊子中微生物的获取

DOI：
10.6084/m9.figshare.19581930
发表时间：
2022
期刊：
影响因子：
0
作者：
Coon K
通讯作者：
Coon K

Inhibition of Protein N-Glycosylation Blocks SARS-CoV-2 Infection.

DOI：
10.1128/mbio.03718-21
发表时间：
2021-02-22
期刊：
mBio
影响因子：
6.4
作者：
Casas-Sanchez A;Romero-Ramirez A;Hargreaves E;Ellis CC;Grajeda BI;Estevao IL;Patterson EI;Hughes GL;Almeida IC;Zech T;Acosta-Serrano Á
通讯作者：
Acosta-Serrano Á

Methods of SARS-CoV-2 Inactivation.

SARS-CoV-2 灭活方法。