Novel synthetic 3'-untranslated regions for controlling transgene expression in transgenic Aedes aegypti mosquitoes

Transgenic technology for mosquitoes is now more than two decades old, and a wide array of control sequences have been described for regulating gene expression in various life stages or specific tissues. Despite this, comparatively little attention has been paid to the development and validation of other transgene-regulating elements, especially 3MODIFIER LETTER PRIME-untranslated regions (3MODIFIER LETTER PRIMEUTRs). As a consequence, the same regulatory sequences are often used multiple times in a single transgene array, potentially leading to instability of transgenic effector genes. To increase the repertoire of characterized 3MODIFIER LETTER PRIMEUTRs available for genetics-based mosquito control, we generated fifteen synthetic sequences based on the base composition of the widely used SV40 3MODIFIER LETTER PRIMEUTR sequence, and tested their ability to contribute to the expression of reporter genes EGFP or luciferase. Transient transfection in mosquito cells identified nine candidate 3MODIFIER LETTER PRIMEUTRs that conferred moderate to strong gene expression. Two of these were engineered into the mosquito genome through CRISPR/Cas9-mediated site-specific insertion and compared to the original SV40 3MODIFIER LETTER PRIMEUTR. Both synthetic 3MODIFIER LETTER PRIMEUTRs were shown to successfully promote transgene expression in all mosquito life stages (larva, pupa and adults), similar to the SV40 3MODIFIER LETTER PRIMEUTR, albeit with differences in intensity. Thus, the synthetic 3MODIFIER LETTER PRIMEUTR elements described here are suitable for regulating transgene expression in Ae. aegypti, and provide valuable alternatives in the design of multi-gene cassettes. Additionally, the synthetic-scramble approach we validate here could be used to generate additional functional 3MODIFIER LETTER PRIMEUTR elements in this or other organisms.