Engineered Promoter System Enables High-efficiency Transgenic CRISPR Editing in Malaria Transmitting Mosquito Anopheles sinensis

The CRISPR/Cas9 system deployed through crosses of transgenic lines expressing Cas9 and gRNA facilitates efficient mutagenesis. However, its application in non-model insects remains limited, primarily due to a lack of well-characterized promoters capable of driving robust and stable expression of Cas9 and gRNA . In the malaria mosquito Anopheles sinensis , we evaluated several ovary-biased promoters— Asvasa2, Aszpg , and Asnanos —for driving Cas9 expression. Notably, the Asvasa2 promoter mediated mutagenesis in nearly 60% of G ₀ individuals following microinjection of gRNA ^Aswhite . Among four RNA polymerase III promoters derived from AsU6 genes, AsU6 -1 yielded the highest gRNA transcriptional output, enabling 62% editing efficiency in G ₀ offspring. In addition, hybrid crosses between established transgenic lines demonstrated that the Asvasa2-Cas9 and AsU6 -1- gRNA combination enabled complete germline editing penetrance, where all F ₂ progeny inherited the intended mutations. This work provides a essential genetic toolkit for synthetic biology applications in Anopheles mosquitoes and a scalable framework for engineering other non-model insects.