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）通过蚊子媒介伊蚊传播给人类 埃及人，并对美洲构成重大的健康风险（1，2）。这个第一阶段的项目来自科罗拉多州 大学（CSU; Olson and Black）、密苏里州大学（MU; Franz）和圣母大学（UND; Fraser）将应用基因驱动技术产生Ae.对ZIKV和其他病毒难治的埃及人群体 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 间隔短回文重复序列（CRISPR）-Cas9用于快速，有效，基于遗传的人群 埃及伊蚊的变种。我们的主要目标是：1）应用两种经过验证的抗病毒转基因（AVTG） 靶向ZIKV以最小化或消除Ae. 2）获得转基因Ae. 在CRISPR/Cas9（CC）基因驱动系统的背景下表达AVTG的埃及人，3）显示， 转基因蚊子对ZIKV感染具有稳定的、难治的表型，以及4）验证基因 驱动器（在室内笼研究）通过渐渗AVTG到一个Ae。埃及人口将皈依 群体从易感感染表型到难治性感染表型。此前，奥尔森博士和弗兰兹 基因改造的Ae。稳定表达DENV 2特异性反向重复（IR）RNA的埃及伊蚊（无驱动） 形成dsRNA以触发蚊子中RNAi的小干扰（si）RNA途径（3，4）。弗雷泽博士 开发了表达I组内含子（GrpI）核酶的蚊子细胞系，所述核酶靶向高度保守的 虫媒病毒基因组的区域。他已经开发出了针对所有四种DENV血清型的GrpI核酶 和基孔肯雅病毒（CHIKV;甲病毒属）（6，7）。在这项提案中，我们将首先开发siRNA， 在CC基因驱动的背景下，基于核酶的抗病毒基因靶向ZIKV。我们将比较两个AVTG 研究人员使用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Grpi AVTG基因成功地抑制了载体感受态，并将ZIKV AVTG传播到笼中的哺乳动物群体中。 AE.埃及，我们将使用具有CC驱动系统的其他GrpI AVTG来另外靶向DENV和CHIKV。 CC基因驱动已经被用于用抗-HCV抗体有效地修饰斯氏按蚊的笼群。 病原基因（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，以大大减少或消除Ae。埃及人传播ZIKV的能力。CC-AVTG系统 我们的目标是两个Ae。埃及伊蚊基因组位点（TIMP P4在染色体aem 2 q和3 'UTR上 多聚腺苷酸结合蛋白（臂3q上的PABP）。我们使用β2-微管蛋白启动子来表达Cas9以实现 通过雄性生殖细胞的基因驱动。这将最大限度地提高同源依赖性修复（HDR）， 而非同源末端连接（NHEJ）会损害驱动（9）。这一建议是一个重要步骤 对开发和优化CC驱动器与AVTG在Ae。埃及人。这一努力将成为 在Ae中使用CC驱动器。我们有信心该项目将大大提高人口 替代作为潜在减轻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.