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

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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.