Performance of low-threshold, population replacement gene drives in cage populations of the yellow fever mosquito, Aedes aegypt

Aedes aegypti is the predominant vector for arboviruses including dengue, Zika, and chikungunya viruses, which infect over 100 million people annually. Mosquito population replacement strategies in which pathogen-susceptible mosquitoes in the field are replaced by laboratory-engineered pathogen-resistant strains are novel genetic control measures to inhibit the spread of malaria or arboviral diseases in endemic regions. To suppress arbovirus transmission following this approach, a mosquito strain needs to be transgenically modified to express an antiviral effector molecule which is linked to a gene drive (GD) system to both inhibit viral replication in the mosquito and drive the engineered resistance trait throughout a wild-type population. As a proof-of-concept, we tested the performance of two single locus CRISPR-Cas9 based GD for Ae. aegypti population replacement in small cage populations over 12 generations. Starting from a low release threshold of 1:9 GD bearing males, we observed two GD constructs in which Cas9 was expressed from two different germline promoters, nanos and zpg, to increase in frequency in all discrete, non-overlapping cage populations. By G12, 56-79% of mosquitoes in six cage populations had at least one GD copy. The allele frequencies of the GD increased from <5% at release to >50% post-release by G7 and G10 for the nanos- and zpg-driven Cas9 GD, respectively. Insertion and deletion mutation (indel) frequency was measured for each discrete generation in pooled samples from the six populations harboring GD. We found that populations with Cas9 expression under control of the nanos-promoter accumulated gene drive blocking indels (GDBI) at more than twice the rate of populations harboring the zpg-promoter driven GD. Both GD produced de novo mutations at similar rates, with a difference in selection being the primary cause of greater indel accrual in the nanos-driven GD populations. Our results demonstrate that two single-locus, CRISPR-Cas9-based homing GD located at an intergenic locus exhibit continuous super-Mendelian inheritance in populations of Ae. aegypti. We further analyze the effects of fitness cost on the stability of low-threshold CRISPR/Cas9 based GD in populations of Ae. aegypti. This study demonstrates the feasibility of low-threshold, single-locus Cas9 gene drives for Ae. aegypti population replacement.