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

通过Cas9驱动的种群改造，对埃及伊蚊进行寨卡病毒抗性工程改造

• 批准号: 9889874
• 负责人: KENNETH E OLSON
• 金额: $ 58.36万
• 依托单位: COLORADO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-04-15 至 2022-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9889874
• 关键词: 3' Untranslated Regions; 3-Dimensional; Aedes; Aedes aegypti genome; Alleles; Americas; Anopheles Genus; Antiviral Agents; Arboviruses; Arthropods; Binding Proteins; CRISPR gene drive; CRISPR/Cas technology; Catalytic RNA; Cell Line; Chikungunya virus; Chromosomes; Cleaved cell; Clustered Regularly Interspaced Short Palindromic Repeats; Collection; Colorado; Competence; Containment; Culicidae; DNA cassette; Double-Stranded RNA; Effectiveness; Elements; Engineering; Flavivirus; Foundations; Future; Gene Transfer Techniques; Generations; Genes; Genetic; Genome; Germ Lines; Goals; Guide RNA; Health; Human; Impairment; Infection; Introns; Inverted Repeat Sequences; Knock-in; Mexico; Midgut; Missouri; Modification; Nonhomologous DNA End Joining; Pathway interactions; Pattern; Phase; Phenotype; Poly A; Population; Population Replacements; Prevalence; Publishing; RNA; RNA Interference; Refractory; Resistance; Risk; Serotyping; Single Nucleotide Polymorphism; Single-Stranded Conformational Polymorphism; Site; Small Interfering RNA; System; Testing; Time; Transgenes; Transgenic Organisms; Tubulin; Universities; Vero Cells; Viral Genes; Virus; ZIKV infection; Zika Virus; base; de novo mutation; disorder control; egg; experimental study; field study; gene drive system; genetic testing; in vitro Assay; integration site; male; mosquito-borne; novel strategies; pathogen; population based; promoter; repaired; success; tool; transmission process; urban area; vector; vector competence; vector mosquito; viral RNA; viral resistance

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.

项目摘要。 Zika病毒（Zikv; Flavivivirus）通过蚊子媒介传播给人类 埃及对美洲有重大健康风险（1，2）。科罗拉多州的I期项目 密苏里大学（MU; Franz）和圣母大学（Underre Dame）大学（CSU; Olson and Black）大学（UND; Fraser）将应用基因驱动技术生成AE。埃及人口对Zikv和其他 该载体物种传播的arbovirus。我们的基因驱动方法定期基于聚类 间隔短的短质体重复序列（CRISPR）-CAS9，用于快速，有效，基于遗传学的种群 修改埃及埃及。我们的主要目标是：1）应用两个经过验证的抗病毒反式基因（AVTG） 针对ZIKV的策略可以最大程度地减少AE的ZIKV感染或消融ZIKV感染。埃及，2）产生转基因AE。 在CRISPR/CAS9（CC）基因驱动系统的背景下表达AVTG的Aegypti，3）表明 一般修饰的蚊子具有稳定的，难以耐磨的表型，用于ZIKV感染，4）验证基因 通过将AVTG浸入AE中，驱动（在室内笼研究中）。埃及人口转换 从胜任到难治性感染表型的人口。以前，Drs。奥尔森和弗朗兹有 通常修改的AE。埃及（没有驱动器）稳定表达DENV2特异性，反向重复（IR）RNA 形成dsRNA以触发蚊子中RNAi的小干扰（Si）RNA途径（3，4）。弗雷泽博士有 开发的蚊子细胞系，表达针对高度保守的I内含子（GRPI）核酶 灰烬病毒基因组区域。他已经开发了针对所有四种DENV血清型的GRPI核酶 和chikungunya病毒（chikv;α）（6，7）。在此提案中，我们最初将开发sirna和 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 CC基因驱动的策略以查看哪种最有效地抑制蚊子感染。如果是grpi AVTG基因成功抑制向量能力并将ZIKV AVTG传播到笼子中 Ae。埃及，我们将与CC驱动系统一起使用其他GRPI AVTG来瞄准DENVS和CHIKV。 CC基因驱动器已被用来有效地修改抗链球菌的笼子群 病原体基因（8）。我们有信心我们可以适应这种方法来驱动抗病毒IR RNA和核酶 AVTG基因可大量减少或消融AE。埃及传输ZIKV的能力。 CC-AVTG系统 我们建议将针对两个AE。埃及基因组站点（染色体AEM 2Q和3'UTR上的TIMP P4 聚腺苷酸结合蛋白（ARM 3Q上的PABP）。我们使用β2-微管蛋白启动子表达CAS9以实现 基因驱动穿过男性种系。这将最大化同源性维修（HDR）以保持驱动器 损害驱动器（9）的非理论最终连接（NHEJ）。该提议代表着重要的一步 用AE中的AVTG开发和优化CC驱动器。埃及。这项努力将是基础 在AE中使用CC驱动器。埃及疾病控制的埃及我们充满信心，该项目大大提高了人口 替换是一种潜在减轻Arbovirus传播的工具。