Development of precision genome editing tools in Ae. albopictus for functional genetics and mosquito control technologies

PROJECT SUMMARY The Asian tiger mosquito, Aedes albopictus, is an aggressive human-biting mosquito that is a competent vector of the dengue, chikungunya and Zika viruses as well as multiple native North American encephalitis viruses. The rapid spread of this mosquito from its native Asian range across the globe during the last 30 years and its well- established role as a primary vector of recent outbreaks of both dengue and chikungunya viruses represents an outstanding public health concern. The goal of this proposal is to produce and test a flexible and efficient precision genome editing system based on CRISPR-Cas9 in A. albopictus. We propose to produce multiple transgenic lines with stable germline expression of the Streptococcus pyogenes Cas9 protein (Cas9) driven by native A. albopictus promoters. Our rationale is that endogenously expressed Cas9 leads to higher editing rates, greater technical efficiency and dramatically decreased costs of genome editing relative to injection of exogenous sources of Cas9. This rationale is supported by our previous work in another closely related vector, Aedes aegypti, where the production of endogenous Cas9 lines not only improved functional genetic capabilities in this vector, but also provided the foundation for the development of genetic-based control technologies. In aim 1, we will produce transgenic lines with native A. albopictus promoter sequences driving Cas9 expression and fluorescent reporters. These lines will be rationally designed based on recent “omics” data in A. albopictus and our extensive work in another closely related vector. Lines will be tested that have different Cas9 promoters and insertion sites and lines with the most robust and stable Cas9 expression will be more extensively evaluated in single and multi-gene knockout studies. In aim 2, we will design and test small guide RNAs to target multiple genes of potential relevance to A. albopictus control including potential phenotypic markers for quality control and sex sorting applications, sex determinate genes and flight specific genes for sterile insect techniques and gene drive applications as well as essential genes of interest for gene drives. The research described in this proposal will advance A. albopictus genomics research by: (1) establishing a platform for rapid and efficient functional genetics and reverse-genetic screens of genes affecting a wide range of phenotypes relevant to disease transmission and vector control, and (2) completing a necessary first step to develop novel tools for vector control such as genetic sexing and gene drive.