Engineering resistance to Zika virus in Aedes aegypti for Cas9 driven population modification

项目摘要。寨卡病毒(ZIKV；黄病毒)通过蚊媒伊蚊传播给人类 埃及伊蚊对美洲构成重大的健康风险(1，2)。科罗拉多州的这一第一阶段项目 密苏里大学(密苏里大学；弗朗茨)和圣母大学(UND； Fraser)将应用基因驱动技术来产生Ae。埃及伊蚊种群对寨卡病毒和其他 虫媒病毒通过这种媒介传播的虫媒病毒。我们的基因驱动方法是基于规则聚类的 间隔短回文重复序列(CRISPR)-CAS9，用于快速、有效、基于遗传学的种群 埃及伊蚊的变种。我们的主要目标是：1)应用两个有效的抗病毒反义基因(AVTG) 针对寨卡病毒的策略，以最大限度地减少或消除寨卡病毒感染Ae。埃及，2)产生转基因Ae。 在CRISPR/Cas9(CC)基因驱动系统中表达AVTG的埃及人，3)表明 转基因蚊子对ZIKV感染具有稳定的、难治的表型，并且4)验证基因 通过将AVTG导入Ae来驾驶(在室内笼子研究中)。埃及伊蚊种群将把 人群从可胜任的感染表型转变为难治感染表型。在此之前，奥尔森博士和弗朗茨博士 转基因Ae.稳定表达DENV2特异性反向重复(IR)RNA的埃及油菜(无驱动) 在蚊子中形成dsRNA以触发RNAi的小干扰(Si)RNA途径(3，4)。弗雷泽博士有 已开发的蚊子细胞株能够表达针对高度保守的GRPI核酶 虫媒病毒基因组的区域。他已经开发出针对所有四种DENV血清型的GrpI核酶 和基孔肯雅病毒(CHIKV；甲型病毒)(6，7)。在这项提案中，我们将首先开发siRNA-和 以核酶为基础的抗病毒基因在CC基因驱动背景下靶向ZIKV。我们将比较这两个AVTG 采用CC基因驱动的策略，看看哪种策略在抑制蚊子感染方面最有效。如果这组人 AVTG基因成功地抑制了载体能力，并将ZIKV AVTG传播到笼养种群中 天哪。Aegypti，我们将使用其他GrpI AVTG和CC驱动系统来额外瞄准DENV和CHIKV。 CC基因驱动已被用于对斯氏按蚊笼养种群进行高效的抗病毒修饰。 致病基因(8)。我们相信，我们可以采用这种方法来驱动抗病毒IR、RNA和核酶 AVTG基因可显著降低或消融Ae。埃及伊蚊传播寨卡病毒的能力。CC-AVTG系统 我们建议将目标对准两个Ae。埃及伊蚊基因组位点(染色体AEM 2Q和3‘UTR上的TIMP P4 多聚腺苷结合蛋白(PABP On Arm 3Q)。我们使用β2-微管蛋白启动子来表达Cas9，以实现 基因驱动着雄性生殖系。这将最大限度地提高同源依赖修复(HDR)以保持驱动力 过度非同源末端连接(NHEJ)，这会损害驱动力(9)。这项提议是迈出的重要一步。 开发和优化Ae中带有AVTGs的CC驱动器。埃及伊蚊。这一努力将为 在Ae中使用CC驱动器。埃及伊蚊用于疾病控制我们相信该项目将极大地提高人口数量 替代作为潜在缓解虫媒病毒传播的工具。

Project Summary. Zika virus (ZIKV; Flavivirus) is transmitted to humans by the mosquito vector Aedes aegypti and poses significant health risks to the Americas (1, 2).This phase I project from Colorado State University (CSU; Olson and Black), University of Missouri (MU; Franz) and University of Notre Dame (UND; Fraser) will apply gene drive technology to generate Ae. aegypti populations refractory to ZIKV and other arboviruses transmitted by this vector species. Our gene drive approach is based on Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 for rapid, effective, genetics-based population modification of Aedes aegypti. Our principal goals are: 1) apply two validated Anti-Viral Trans Gene (AVTG) strategies that target ZIKV to minimize or ablate ZIKV infection of Ae. aegypti, 2) generate transgenic Ae. aegypti that express the AVTGs in the context of a CRISPR/Cas9 (CC) gene drive system, 3) show that the genetically modified mosquitoes have a stable, refractory phenotype for ZIKV infection, and 4) validate gene drive (in indoor cage studies) by introgressing the AVTGs into an Ae. aegypti population to convert the population from a competent to a refractory infection phenotype. Previously, Drs. Olson and Franz have genetically modified Ae. aegypti (with no drive) that stably express DENV2-specific, inverted repeat (IR) RNAs forming dsRNA to trigger the small interfering (si)RNA pathway of RNAi in the mosquito (3, 4). Dr. Fraser has developed mosquito cell lines that express group I intron (GrpI) ribozymes targeted to a highly conserved region of arboviral genomes. He has already developed GrpI ribozymes that target all four DENV serotypes and chikungunya virus (CHIKV; Alphavirus) (6, 7). In this proposal, we will initially develop siRNA- and ribozyme-based anti-viral genes to target ZIKV in the context of CC gene drive. We will compare the two AVTG strategies with CC gene drive to see which is the most effective at suppressing mosquito infection. If the GrpI AVTG genes successfully suppress vector competence and spread the ZIKV AVTG into caged populations of Ae. aegypti, we will use other GrpI AVTGs with the CC drive system to additionally target DENVs and CHIKV. CC gene drive has already been used to efficiently modify cage-populations of Anopheles stephensi with anti- pathogen genes (8). We are confident we can adapt this approach to drive antiviral IR RNA and ribozyme AVTG genes to greatly reduce or ablate Ae. aegypti competence for transmitting ZIKV. The CC-AVTG system we propose will target two Ae. aegypti genome sites (TIMP P4 on chromosome aem 2q and 3'UTR polyadenylate binding protein (PABP on arm 3q). We use the β2-tubulin promoter to express Cas9 to achieve gene drive through the male germ-line. This will maximize homology-dependent repair (HDR) to maintain drive over non-homologous end-joining (NHEJ) which impairs drive (9). This proposal represents a significant step towards developing and optimizing CC drive with AVTGs in Ae. aegypti. This effort will be foundational for using CC drive in Ae. aegypti for disease control We are confident the project greatly advances population replacement as a tool for potentially mitigating arbovirus transmission.