Optimization of low-threshold Cas9-based gene drive systems to introduce Zika virus resistance in Aedes aegypti

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Project Summary Aedes aegypti is the principal vector of arthropod-borne viruses (arboviruses) affecting human health in the tropics, such as Zika, chikungunya, and dengue viruses. Zika virus (ZIKV) caused the most recent major epidemic arbovirus outbreak in the New World Hemisphere. As a wide-spread, peridomestic vector in tropical regions, Ae. aegypti has become ever more difficult to control. Many populations have become resistant to commonly used insecticides increasing the need for alternative control strategies. One such alternative is the use of genetically-modified virus-resistant mosquitoes to disrupt the arboviral disease cycle, however this approach requires a mechanism to spread and fix the transgene within a population. Newly developed gene drive (GD) systems are capable of this, as they bias the inheritance of genes in a super-Mendelian fashion. GD systems can be broadly classified into population suppression and population replacement, with the former resulting in population collapse, and the latter resulting in the fixation of a transgene in a population. Population replacement is a concept still under development, and requires two genetic components: a strong antiviral effector, and a robust GD system. Due to the GD-linked spread of the anti-viral effector gene, a targeted virus- susceptible wild population would be converted into a virus-resistant one. Recently, we developed an anti-viral effector gene that specifically targets ZIKV by triggering the mosquito’s RNAi pathway. Here, we seek to combine the anti-ZIKV effector with low-threshold homing GDs based on Cas9, which so far, have not been developed for Ae. aegypti. These GDs rely on homology dependent DNA repair in the germline, however the expression of Cas9 outside of the germline can lead to GD-resistant indels. GD-blocking indels can be mitigated through the optimization of Cas9 expression, or through the use of novel GD architectures capable of removing such indels when they arise. In four Specific Aims (SA), we propose to develop for Ae. aegypti reliable low-threshold GD systems which are linked to an anti-ZIKV effector: 1) Identify the optimal promoter for Cas9 expression in single-component GD at different genomic loci; 2) Design and generate novel GD (ClvR, HomeR) variants that remove indels; 3) Develop germline::Cas9 gene fusions for a dual-GD systems to allow for the independent drive of the anti-ZIKV effector; 4) Monitor the activity of the optimized GD systems (SA.1-3) in wild-type like mosquitoes via non-overlapping small cage studies over multiple generations. The four SA of this application will identify a GD design for Ae. aegypti that will allow for the robust introduction of new traits, such as resistance to ZIKV, into wild mosquito populations.