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

## TL;DR

Researchers tested two CRISPR-based gene drives in mosquitoes to replace wild populations with virus-resistant ones, showing they can spread effectively but with differing mutation rates.

## Contribution

Demonstrated the performance of two low-threshold CRISPR/Cas9 gene drives in Aedes aegypti cage populations, highlighting the impact of promoter choice on mutation rates and spread efficiency.

## Key findings

- Both gene drives increased in frequency over 16 generations, with 72% and 82% of mosquitoes carrying at least one GD copy.
- The nanos-GD accumulated gene drive blocking indels at more than twice the rate of the zpg-GD.
- Simulations showed the zpg-GD led to more robust population replacement than the nanos-GD.

## Abstract

Aedes aegypti is the predominant vector for arboviruses including dengue, Zika, and chikungunya viruses, which infect over 100 million people annually. Mosquito population replacement in which arbovirus-susceptible mosquitoes in the field are replaced by laboratory-engineered refractory mosquitoes represents a novel genetic control measure to interrupt arboviral disease cycles. For this approach, the engineered mosquitoes need to harbor two genetic components: an antiviral effector construct which is linked to a gene drive (GD). We tested the performance of two single-locus CRISPR/Cas9 based GD for Ae. aegypti population replacement in small cage populations for up to 16 generations. Starting from a low release threshold of 1:9 GD bearing males to wild-type 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 cage populations. By G16, an average of 72% and 82% of individuals from the zpg-GD and nanos-GD populations, respectively, harbored at least one GD copy with corresponding increases in allele frequencies. This indicated that the two single-locus, CRISPR/Cas9-based homing GD exhibited continuous super-Mendelian inheritance in populations of Ae. aegypti. Gene drive blocking indel (GDBI, a.k.a. “resistant alleles”) 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 GDBI at more than twice the rate of those populations harboring the zpg-promoter driven GD. Based on preexisting data sets for homing and GDBI frequencies in addition to the cage trial observations, the relative contributions of sex-specific homing rates, maternal Cas9 deposition and potential fitness effects were modeled in MGDrivE for both GD, further explaining their divergent performance. Our study demonstrates the feasibility of low-threshold, single-locus CRISPR/Cas9 based GD for Ae. aegypti population replacement.

The mosquito Aedes aegypti is the major tropical vector for important human-pathogenic arboviruses. A novel genetic approach to suppress arbovirus transmission in the field includes population replacement with engineered virus-resistant mosquitoes. This approach requires the engineered mosquitoes to harbor two genetic components: 1) a potent antiviral effector that is 2) tightly linked to a gene drive (GD) system. Here we assess the performance of two single-locus, low-threshold GD in generational cage populations. Our data show that the two GD, which differ only by their germline specific promoter driving Cas9 expression, nanos or zpg, facilitated similar population invasion from low-threshold release conditions. However, in the cage populations, the nanos-GD generated more gene drive blocking indels (GDBI) over time than the zpg-GD. In continuous stochastic simulations, the zpg-GD resulted in a more robust population replacement than the nanos-GD. These results illustrate the importance of not just the homing frequency of a GD but also its GDBI formation rate, both of which strongly affect the stability of a GD in population replacement applications. Overall, our results suggest that autonomous, single-locus CRISPR/Cas9 based GD can be suitable vehicles for the sustained spread of antiviral effector transgenes in naïve populations of Ae. aegypti.