Understanding the resistance landscape to gene drives targeting ultra-conserved regions in the doublesex gene of Anopheles gambiae

蚊子是地球上最致命的动物，每年造成75万人死亡，其中50万人死于疟疾传播。在当地限制疟疾传播的最有效方法是使用杀虫剂。随着杀虫剂抗药性的上升，需要采取替代办法来对付主要的疟疾病媒冈比亚按蚊。基因驱动是自私的遗传元素，有可能对整个种群进行遗传修饰或抑制，因为它们可以在种群内以自我维持的方式传播，尽管会产生适应性成本。最近，采用CRISPR/Cas9技术的基因驱动器成功地用于通过靶向doublemex基因的雌性特异性外显子来消除疟疾蚊子的笼中种群。这导致纯合的雌性发展为不育的雌雄同体个体，最终导致笼中种群崩溃。然而，阻止基因驱动活性但编码doubletex的功能性拷贝的靶位点抗性等位基因可以阻止基因驱动在野生环境中的传播。抗性等位基因可以是天然存在的或由基因驱动本身产生的。为了评估double-ex的耐药性并揭示该位点可耐受的核苷酸多样性的量，我使用了两种基于基因驱动靶位点的定向进化和位点特异性诱变的方法。这两种策略可以一起用于在实验室或现场测试之前评估任何基因驱动。在第一种方法中，基因驱动靶位点暴露于重复的Cas9切割，以迫使末端连接突变的产生。为了确定这些突变是否可以恢复doubletex基因功能，我评估了女性的性发育。通过进行这个实验，我也了解了末端连接修复产生的突变的类型和频率。在第二种方法中，我使用CRISPR/Cas9技术对基因驱动靶位点进行靶向诱变，将其替换为该基因座的天然变体，该基因座在野生型中的频率较低。冈比亚种群或邻近的按蚊物种。我的目的是测试鉴定的变体是否成功地阻止基因驱动切割，同时恢复doubletex功能。如果是这样的话，那么它们可能会阻止基因驱动在野外的传播。为了减少在野外选择耐药性的可能性，我建立了一个基因驱动器，它同时靶向doubletex上的两个位点，这是一种类似于联合药物治疗的策略。初步测试表明，新的基因驱动器成功地切割了两个位点，并且比以前针对单个位点的版本表现更好。然而，它仍有待于在实验室环境中进行种群入侵实验。

Mosquitoes are the deadliest animals on earth, causing 750K deaths annually, of which 500K are attributed to malaria transmission. The most effective ways to locally limit malaria spread are based on the use of insecticides. With insecticide resistance on the rise, there is need for alternative approaches to tackle the major malaria vector, Anopheles gambiae. Gene drives are selfish genetic elements with the potential to genetically modify or suppress entire populations, as they can spread in a self-sustaining way within the population, despite conferring a fitness cost. Recently, a gene drive employing the CRISPR/Cas9 technology was successfully used to eliminate caged populations of the malaria mosquito by targeting the female-specific exon of the doublesex gene. This caused females that were homozygous for the drive to develop as sterile intersex individuals, eventually causing caged populations to crash. However, target site resistant alleles that prevent gene drive activity, but encode a functional copy of doublesex may halt gene drive spread in the wild. Resistant alleles may be naturally occurring or generated by the gene drive itself. To evaluate the resistance landscape at doublesex and reveal the amount of nucleotide diversity that can be tolerated at that site, I am using two approaches based upon directed evolution and site-specific mutagenesis of the gene drive target site. Together both strategies can be used to assess any gene drive prior to laboratory or field testing. In the first approach the gene drive target site is exposed to repeated Cas9 cleavage, to force the generation of end-joining mutations. To determine whether these mutations can restore doublesex gene function, I assess the sexual development of females. By performing this experiment, I am also gaining insight in the types and frequency of mutations generated by end-joining repair. In the second approach, I am using the CRISPR/Cas9 technology to perform targeted mutagenesis of the gene drive target site,replacing it with natural variants of this locus, found at low frequencies in wild in An. gambiae populations or in neighbouring Anopheles species. I aim to test whether identified variants successfully prevent gene drive cleavage, whilst restoring doublesex function. If this is the case then they could potentially halt gene drive spread in the wild. To mitigate the likelihood of resistance being selected in the wild, I have built a gene drive that targets two sites on doublesex simultaneously, in a strategy akin to combination drug therapy. Preliminary testing shows that the novel gene drive successfully cuts both sites and performs better than previous versions targeting a single site. However it remains to be tested in population invasion experiments in a laboratory context.