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

Hong, J.-F.; Zou, Q.-L.; Xie, X.-Y.; Jiang, Y.-P.; Wang, S.-Y.; Ling, X.; Zhou, C.; Sun, W.; Cai, X.; Yang, Y.-X.; Chen, Y.; Chen, B.; Qiao, L.

doi:10.1101/2025.09.29.679135

bioRxiv · 10.1101/2025.09.29.679135

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

Hong, J.-F.·Zou, Q.-L.·Xie, X.-Y.·Jiang, Y.-P.·Wang, S.-Y.·Ling, X.·Zhou, C.·Sun, W.·Cai, X.·Yang, Y.-X.·Chen, Y.·Chen, B.·Qiao, L.

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Abstract

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 G0 individuals following microinjection of gRNAAswhite. Among four RNA polymerase III promoters derived from AsU6 genes, AsU6-1 yielded the highest gRNA transcriptional output, enabling 62% editing efficiency in G0 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 F2 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.

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Hong, J.-F., Zou, Q.-L., Xie, X.-Y., Jiang, Y.-P., Wang, S.-Y., Ling, X., Zhou, C., Sun, W., Cai, X., Yang, Y.-X., Chen, Y., Chen, B., Qiao, L.. 2025-09-30. Engineered Promoter System Enables High-efficiency Transgenic CRISPR Editing in Malaria Transmitting Mosquito Anopheles sinensis. https://doi.org/10.1101/2025.09.29.679135

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