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.

摘要：迄今为止，疟疾和其他媒介传播的疾病给人类带来了巨大的负担。 阻止导致疟疾的疟原虫传播的运动依赖于分发 治疗感染者的方法，以及使用浸有杀虫剂的蚊帐和室内滞留喷雾剂的药物 阻止按蚊传播感染。历史上以蚊子为目标。 这些基于杀虫剂的方法是我们控制疾病传播的最佳武器，但是 蚊子种群正以惊人的速度对杀虫剂产生抗药性，使得疾病控制变得困难 寻找旨在控制疟疾传播的新的强有力策略变得越来越具有挑战性。 按蚊种群，我们现在可以在这个项目中利用新型强大的基因组工程工具。 在冈比亚按蚊中使用 CRISPR/Cas 技术来研究蚊子的关键方面 基础生物学并实现新一代遗传控制策略。 在我的研究期间，我验证了 CRISPR/Cas 在冈比亚疟原虫中的功能，并开发了一套强大的 我将使用基因工程工具来研究繁殖和矢量能力之间的新型串扰， 以及生成和测试用于种群抑制和替代的新型遗传控制策略。 使用 CRISPR，我在零种群增长中产生了一系列具有大量缺失的突变蚊子 (ZPG)，一种对生殖细胞发育至关重要的基因，由此产生的雌性突变体的卵巢萎缩，而雄性突变体的卵巢萎缩。 在恶性疟原虫（最致命的疟疾）的感染实验中显示没有精子。 寄生虫，无法发育卵的雌性受到寄生虫的感染较少，这表明两者之间存在联系 因此，在本提案中，我的目的是阐明其作用。 此外，我的目标是探索恶性疟原虫发育中基于卵巢的信号传导的潜力。 ZPG 突变无精子雄性将用于昆虫不育技术（SIT）以抑制种群数量 活动（目标 1B）。 CRISPR/Cas 还可用于促进基因驱动系统能够将所需性状固定在 利用我在这项技术方面的专业知识，在目标 2 中，我将开发一种“进化”的方法。 稳定的基因驱动系统可以强有力地传播所需的性状，以促进对抗疟疾。 系统将通过针对冈比亚疟原虫中聚集在一起的两个必需基因来保证驱动传播 基因组，使得不正确的驱动器复制不可行，此外，该系统将可以轻松编辑以进行测试。 各种驱动架构和不同的抗疟疾或消毒货物。 该项目的研究结果将有助于扩大我们对蚊子生物过程的了解 塑造矢量能力，并将扩大可用于操纵野生按蚊的遗传工具包 人口。