Using genome engineering to study mosquito biology and combat malaria

利用基因组工程研究蚊子生物学并对抗疟疾

• 批准号: 9192424
• 负责人: Andrea Lynelle Smidler
• 金额: $ 3.66万
• 依托单位: HARVARD SCHOOL OF PUBLIC HEALTH
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-11-01 至 2018-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9192424
• 关键词: Affect; Africa; Alleles; Anopheles Genus; Anopheles gambiae; Antimalarials; Architecture; Atrophic; Behavior; Biological Process; Biology; Bite; CRISPR/Cas technology; Child; Clustered Regularly Interspaced Short Palindromic Repeats; Culicidae; DNA Double Strand Break; Development; Disease; Docking; Engineering; Enzyme-Linked Immunosorbent Assay; Essential Genes; Female; Female Sterilization; Gap Junctions; Gene Cluster; Gene Transfer Techniques; Generations; Genes; Genetic; Genetic Engineering; Genetic Transcription; Genome; Genome engineering; Germ Cells; Guide RNA; Infection; Insecta; Insecticides; Knock-in; Knock-out; Knowledge; Lead; Link; Malaria; Methods; Molecular; Mosquito Control; Mutagenesis; Ovarian; Ovary; Parasites; Partner in relationship; Pathway interactions; Plasmodium; Plasmodium falciparum; Population; Property; Reproduction; Reproductive Biology; Residual state; Resistance; Resistance development; Role; Shapes; Signal Transduction; Site; Specificity; Sterility; Sterilization; System; Techniques; Technology; Testing; Testis; Vaccines; Vector-transmitted infectious disease; Zero Population Growth; base; combat; disorder control; drug distribution; egg; fight against; genetic approach; genetic element; genome editing; homologous recombination; innovation; insight; killings; knockout gene; malaria infection; malaria transmission; male; mutant; next generation; novel; nuclease; prevent; programs; promoter; reproductive; research study; sample fixation; sperm cell; tool; trait; transcriptome sequencing; transmission process; vector; vector control; vector mosquito; weapons

ABSTRACT. Malaria and other vector-borne diseases pose an immense burden on mankind. To date, control campaigns to stop transmission of the Plasmodium parasites that cause malaria have relied on the distribution of drugs to treat those infected, and on the use of insecticide-impregnated bednets and indoor residual sprays to stop Anopheles mosquitoes from transmitting the infection. Historically targeting the mosquito vector with these insecticide-based methods has been our best weapon for controlling the spread of the disease, but mosquito populations are developing resistance to insecticides at an alarming rate, making disease control increasingly challenging. In the search for new powerful strategies aimed at controlling malaria-transmitting Anopheles populations, we can now exploit novel powerful genome engineering tools. In this project I am to use CRISPR/Cas technology in Anopheles gambiae to enable studies into critical aspects of mosquito basic biology and to enable a new generation of genetic control strategies. During my studies I have validated the function of CRISPR/Cas in A. gambiae and developed a powerful set of genetic engineering tools that I will use to study a novel crosstalk between reproduction and vectorial capacity, as well as to generate and test novel genetic control strategies for population suppression and replacement. Using CRISPR I have generated a line of mutant mosquitoes with large deletions in Zero Population Growth (ZPG), a gene critical for germ cell development. Resulting female mutants have atrophied ovaries while males show no sperm in the testes. In infection experiments with Plasmodium falciparum, the most deadly malaria parasite, females that are unable to develop eggs become less infected with parasites, suggesting a link between signaling from the ovaries and Plasmodium development. Therefore in this proposal I aim to elucidate the role of ovary-based signaling on P. falciparum development (Aim 1A). Furthermore I aim to explore the potential for the ZPG mutant spermless males to be used in Sterile Insect Technique (SIT) for population suppression campaigns (Aim 1B). CRISPR/Cas can also be used to facilitate gene drive systems capable of spreading desired traits to fixation in natural mosquito populations. Using my expertise in this technology, in Aim 2 I will develop an ‘evolutionarily stable’ gene drive system to robustly spread desirable traits to facilitate the fight against malaria. This drive system will guarantee drive spread by targeting two essential genes clustered together in the A. gambiae genome, making incorrect drive copying inviable. Further the system will be easily editable to enable testing of a wide variety of drive architectures and different anti-malarial or sterilizing cargoes. The findings of this project will be instrumental for expanding our knowledge of mosquito biological processes shaping vectorial capacity, and will expand the genetic toolkit available for the manipulation of wild Anopheles populations.

抽象的。疟疾和其他媒介传播疾病对人类造成了巨大的伯宁。迄今为止，控制 停止导致疟疾的疟原虫寄生虫传播的运动 治疗受感染者的药物，以及使用杀虫剂浸渍的床头和室内残留喷雾剂 阻止蚊子传播感染。从历史上看，用 这些基于杀虫剂的方法一直是我们控制疾病传播的最佳武器，但是 蚊子种群正在以惊人的速度发展对杀虫剂的抵抗力，使疾病控制 越来越挑战。在寻找旨在控制疟疾传播的新的强大策略中 肛门种群，我们现在可以利用新颖的强大基因组工程工具。在这个项目中，我要 在Anopheles Gambiae中使用CRISPR/CAS技术，使研究能够研究蚊子的关键方面 基本生物学并实现新一代的遗传控制策略。 在研究期间，我验证了A. Gambiae中CRISPR/CAS的功能，并开发了一组强大的 我将使用的基因工程工具来研究繁殖和媒介能力之间的新型串扰， 以及生成和测试新的遗传控制策略，以抑制人群和替代。 使用CRISPR，我产生了一系列突变蚊子，其中大量删除在零人口增长中 （ZPG），一种对生殖细胞发育至关重要的基因。由此产生的女性突变体具有萎缩的卵巢 在测试中没有显示精子。在恶性疟原虫的感染实验中，最致命的疟疾 寄生虫，无法发育卵的雌性被寄生虫感染较少，这表明 来自卵巢和疟原虫发育的信号传导。因此，在此提案中，我旨在阐明角色 基于卵巢的恶性疟原虫发育（AIM 1A）的信号。此外，我的目标是探索潜力 ZPG突变的无精子雄性用于无菌昆虫技术（SIT）进行种群抑制 广告系列（AIM 1B）。 CRISPR/CAS还可以用于促进能够传播所需特征以固定的基因驱动系统 天然蚊子种群。利用我在这项技术方面的专业知识，在AIM 2中，我将在进化上发展一个 稳定的基因驱动系统可牢固地传播理想的特征，以促进与疟疾的斗争。这个驱动器 系统将通过靶向两个基本基因聚集在gambiae中来确保驱动传播 基因组，使驱动器复制不正确。此外，该系统将很容易编辑以实现测试 各种各样的驱动架构以及不同的反车道或消毒货物。 该项目的发现将有助于扩大我们对蚊子生物学过程的了解 塑造矢量能力，并将扩大可用于操纵野生动物的遗传工具包 人群。