# Monoallelic knockout of r2d2 affects the antiviral RNAi response to Mayaro virus and the reproductive potential in Aedes aegypti

## TL;DR

This study shows that knocking out the r2d2 gene in Aedes aegypti mosquitoes affects their antiviral response and reproduction, with monoallelic disruption increasing Mayaro virus replication.

## Contribution

The study reveals the essential role of r2d2 in RNAi and reproduction, and how its partial loss affects viral infection and compensatory piRNA activity.

## Key findings

- Complete loss of r2d2 is lethal in Aedes aegypti, preventing homozygous knockout.
- Monoallelic r2d2 disruption increases Mayaro virus replication and vpiRNA abundance.
- R2D2 is essential for RISC loading and siRNA pathway function, but dicer-2 remains unaffected.

## Abstract

Aedes aegypti is an important vector for several human-pathogenic arboviruses. RNAi is the principal antiviral immune pathway in mosquitoes. Key steps of antiviral RNAi are processing of long dsRNAs into siRNA duplexes by dicer-2; loading of the siRNA duplexes onto Argonaute-2 with the help of R2D2; RISC formation via incorporation of Argonaute-2, which contains an siRNA; RISC-mediated targeting and degradation of homologous viral RNAs. Here, we generated an r2d2 knockout mosquito line to reveal how RNAi impairment during RISC loading complex (RLC) formation would affect arbovirus infection of Ae. aegypti.

CRISPR/Cas9 gene editing has been used to knock out r2d2 in Ae. aegypti. Crossing experiments were conducted to reveal the effects of loss of r2d2 function on fecundity and fertility. Mayaro virus (Togaviridae: MAYV) infection and RNAi pathway gene expression levels were monitored using time-course RT-qPCR assays. Small RNA profiling was conducted to determine small RNA abundance in ΔR2D2(+/-) mosquitoes.

We show that in Ae. aegypti, the r2d2 allele is linked to the sex determination locus on chromosome 1. It was not possible to generate homozygous ΔR2D2(−/−) mosquitoes, indicating that complete loss of r2d2 function is lethal to Ae. aegypti. Our observations suggest that r2d2 function is not limited to RNAi but also affects mosquito fecundity/fertility, likely through follicle development. Monoallelic disruption of r2d2 increased the replication of MAYV, and r2d2 expression was also increased in infected mosquitoes. MAYV infection of ΔR2D2(+/-) mosquitoes was associated with an increase in abundance of putative vpiRNAs. However, impairment of r2d2 did not affect the function of dicer-2, as there was no difference in the 21 nt siRNA profiles between the ΔR2D2(+/-) mosquitoes and the non-transgenic control.

The RNAi pathway gene, r2d2, is an essential gene, and it is not possible to generate mosquitoes with biallelic (complete) loss of r2d2 function. Monoallelic impairment of r2d2 compromises the siRNA pathway downstream of dicer-2 function, at the point of RLC formation. In MAYV-infected mosquitoes, this defect in siRNA pathway function is compensated for by an increased piRNA pathway activity, which moderates increases in viral replication over a 10-day period.

The online version contains supplementary material available at 10.1186/s13071-026-07265-9.