Highly efficient Cas9-mediated gene drive for population modification of the malaria vector mosquito Anopheles stephensi
Highly efficient Cas9-mediated gene drive for population modification of the malaria vector mosquito Anopheles stephensi
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DOI：
10.1073/pnas.1521077112
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发表时间：
2015-12-08
影响因子：
11.1
通讯作者：
James, Anthony A.
中科院分区：
文献类型：
--
作者：
Gantz, Valentino M.;Jasinskiene, Nijole;James, Anthony A.
基因工程技术既可用于创造携带针对人类疟疾寄生虫的抗病原体效应基因的转基因蚊子，也可用于产生能够将基因渗入整个野生载体种群的基因驱动系统。我们在亚洲疟疾载体斯氏按蚊中开发了一种高效自主的规则间隔短回文重复序列（CRISPR）相关蛋白9（Cas9）介导的基因驱动系统，该系统改编自诱变链反应（MCR）。该特定系统导致源自转基因雄性的雄性和雌性的后代表现出与同源定向修复（HDR）一致的高频率的生殖系基因转换。该系统复制一个类似于17 kb的结构，从其插入位点到其同源染色体上，在一个忠实的，位点特异性的方式。双抗恶性疟原虫效应基因，标记基因，和自主基因驱动组件渗入到类似的99.5%的后代后，异交的转基因系野生型蚊子。效应基因在血液喂养后保持转录诱导。与仅在生殖系中表达Cas9的个体中的有效转化相反，预期在卵中具有驱动组分分子的源自转基因雌性的雄性和雌性产生在靶向基因组序列中具有高频率突变的后代，导致转基因的近孟德尔遗传比。这种突变等位基因可能是在发育早期体细胞和生殖系分离之前由非同源末端连接（NHEJ）事件引起的。这些数据支持该系统的设计严格在生殖系内有效。作为消灭疟疾议程的一部分，基于这种技术的菌株可以持续控制和消灭。
Genetic engineering technologies can be used both to create transgenic mosquitoes carrying antipathogen effector genes targeting human malaria parasites and to generate gene-drive systems capable of introgressing the genes throughout wild vector populations. We developed a highly effective autonomous Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated protein 9 (Cas9)-mediated gene-drive system in the Asian malaria vector Anopheles stephensi, adapted from the mutagenic chain reaction (MCR). This specific system results in progeny of males and females derived from transgenic males exhibiting a high frequency of germ-line gene conversion consistent with homology-directed repair (HDR). This system copies an similar to 17-kb construct from its site of insertion to its homologous chromosome in a faithful, site-specific manner. Dual anti-Plasmodium falciparum effector genes, a marker gene, and the autonomous gene-drive components are introgressed into similar to 99.5% of the progeny following outcrosses of transgenic lines to wild-type mosquitoes. The effector genes remain transcriptionally inducible upon blood feeding. In contrast to the efficient conversion in individuals expressing Cas9 only in the germ line, males and females derived from transgenic females, which are expected to have drive component molecules in the egg, produce progeny with a high frequency of mutations in the targeted genome sequence, resulting in near-Mendelian inheritance ratios of the transgene. Such mutant alleles result presumably from non-homologous end-joining (NHEJ) events before the segregation of somatic and germ-line lineages early in development. These data support the design of this system to be active strictly within the germ line. Strains based on this technology could sustain control and elimination as part of the malaria eradication agenda.