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

> **NIH NIH R56** · UNIVERSITY OF MISSOURI-COLUMBIA · 2022 · $537,024

## Abstract

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.

## Key facts

- **NIH application ID:** 10667935
- **Project number:** 1R56AI167980-01
- **Recipient organization:** UNIVERSITY OF MISSOURI-COLUMBIA
- **Principal Investigator:** Alexander W E Franz
- **Activity code:** R56 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $537,024
- **Award type:** 1
- **Project period:** 2022-08-10 → 2025-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10667935

## Citation

> US National Institutes of Health, RePORTER application 10667935, Optimization of low-threshold Cas9-based gene drive systems to introduce Zika virus resistance in Aedes aegypti (1R56AI167980-01). Retrieved via AI Analytics 2026-05-26 from https://api.ai-analytics.org/grant/nih/10667935. Licensed CC0.

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