MitoPac female killing systems in mosquitoes

PROJECT SUMMARY Dengue, chikungunya, and Zika are diseases transmitted by Aedes aegypti. One strategy to reduce Ae. aegypti populations is to release non-biting male mosquitoes that pass female killing genes to their progeny. Over time and with many releases, this can lead to a significant suppression of the mosquito population, decreasing transmission of the pathogens they vector. Some female-killing technologies are available for this vector, but these technologies have some challenges that support the development of additional control technologies. To address this need, we aim to build new tools to generate female-killing technologies in Ae. aegypti. We focus on developing novel mitochondrial-targeted restriction endonucleases (mtREs) and CRISPR systems to kill females with flexible designs and components amenable for future development in a wide variety of mosquito vectors. As part of these efforts, we will explore switch-like cis-regulatory polycomb response elements (PREs) to modulate the transcriptional activity of the mtREs and Cas9. In Aim 1, we will selectively express the small mtRE, PacI, in female mosquitoes. Female-specific expression of PacI will be achieved by driving PacI expression with a female-specific promoter or by encoding a female-specific intron of the highly conserved doublesex (dsx) gene into the coding sequence of PacI. These expression systems will be further optimized by integrating the PRE upstream of the PacI promoter. In Aim 2, we will also use PREs to develop high precision heat shock-inducible and female-specific Cas9 expression systems. Heat shock promoters can still have activity at low temperatures, and Cas9 often has high activity, which can interfere with strain survival and the desired female-killing phenotype, so we aim to use PREs to minimize the issues. To achieve female specificity, the heat shock Cas9 will be designed to target female-specific essential genes, or the Cas9 will be engineered to encode a female-specific dsx intron in its coding sequence, ensuring Cas9 is only expressed in females. To optimize these Cas9 systems, they will be engineered first to target an easily screenable gene and then to target essential genes. The tools developed in Aims 1 and 2 will be evaluated in preliminary fitness and population cage studies, which will be used to prioritize their future development in Ae. aegypti and other mosquito vectors. These expression systems also have other applications for mosquito control and as a research tool to explore mosquito biology and genetics.