A potent EF1A promoter and a minimal U6 promoter enable highly efficient CRISPR genome editing in Aedes aegypti

CRISPR/Cas9 has become a key tool for functional genomics and genetic control strategies in mosquitoes, with its efficiency highly dependent on precise spatiotemporal regulation of Cas9 expression. While germline-specific promoters help reduce resistance and improve drive efficiency, constitutive promoters confer higher editing efficiency due to their ubiquitous and sustained expression, making them more suitable for gene functional analysis and population-specific suppression. However, the scarcity of strong constitutive promoters in Aedes aegypti (Ae. aegypti) has limited the development of highly efficient editing systems. In this study, we cloned the promoter of the housekeeping gene eukaryotic translation elongation factor 1α (EF1Α, AAEL017096) and confirmed its transcriptional activity. RT-qPCR analysis revealed that the EF1Α promoter (EF1Αp) directed significantly higher Cas9 expression in mosquito ovaries and testes than did the germline-specific Exu promoter (Exup). Crossing EF1Ap-Cas9 transgenic lines with various gRNA-expressing lines achieved efficient editing of the marker gene white, the functional gene PNP, and the microRNA precursor miR-1174, with all double-positive progeny displaying expected loss-of-function phenotypes. To overcome vector capacity limitations, we truncated the U6 promoter. A truncated U6 promoter of only 235 bp (U6p235) supported editing efficiency comparable to the full-length 964 bp version. When U6p235-white gRNA lines were crossed with EF1Ap-Cas9 lines, all double-positive offspring exhibited a white-eye phenotype, confirming successful white gene disruption. Together, this work provides an efficient constitutive promoter and a compact U6p, expanding the genetic toolkit for Ae. aegypti and supporting advanced functional studies and multi-target control strategies.