A Tailor-made CRISPR Suit for the "Asian Tiger Mosquito" Aedes albopictus

Aedes albopictus is an invasive and aggressive mosquito species that pose an increasing global public health concern. To facilitate the development of new control means against this invader, we have tailored aCRISPR/Cas system toolbox using the mosquito endogenous promoters. We looked into Aedes albopictus genome assembly and transcriptomics data to identify and isolate nine pol-II and six pol-III endogenous promoters to enable somatic, as well as germline, Cas9 activity. By constructing strains expressing Cas9 and sgRNA, we began crossing the different lines and screening the different combinations output. By targeting the yellow gene, we have established a mosaicism assessment method for tracking CRISPR activity and identifying potent combinations, successfully confirming the activity of five out of six pol-III promoters. We showed how this ‘split design’ mechanism can be later used to generate loss-of-function mutations for any gene of interest. These basic building blocks of the CRISPR system can be now used as the foundations for increasing gene editing capabilities, expanding the repertoire of available mutants and the constructions of novel synthetic homing genes.