Abstract
Current methods for gene editing in insects rely on embryonic microinjection, which can be challenging for non-specialist laboratories. Recently, an alternative method known as “direct parental” CRISPR (DIPA-CRISPR) was developed. This method involves injecting commercial Cas9 protein and single-guide RNA into adult females, which can efficiently introduce mutations into developing oocytes. However, its versatility has not been fully explored, particularly in insects that have the most derived, polytrophic meroistic ovaries. In this study, we successfully applied DIPA-CRISPR to the yellow fever mosquito Aedes aegypti, which has polytrophic meroistic ovaries. Following adult injection of Cas9 ribonucleoproteins (Cas9 RNPs) targeting the kynurenine 3-monooxygenase gene, we recovered gene-edited G0 individuals. Injection at 24 h after blood-feeding resulted in the highest gene editing efficiency (3.5%), confirming that a key parameter of DIPA-CRISPR is the stage in which the adult females are injected. Together with our previous study, we demonstrated that DIPA-CRISPR is applicable to all three types of insect ovaries (i.e., panoistic, telotrophic, and polytrophic), which indicates that DIPA-CRISPR is a generalizable approach for insect gene editing.
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Acknowledgements
We thank Takahiro Ohde, Toshiya Ando, and the laboratory members for helpful suggestions and discussion, and Misaki Masuda for assistance with mosquito rearing. This research was supported by JSPS KAKENHI (nos. 20K21311and 22K19179 to T.D.) and Moonshot Research and Development Program for Agriculture, Forestry, and Fisheries (Funding Agency: Bio-oriented Technology Research Advancement Institution) (no. JPJ009237 to T.D. and M.O.). Y.S. is a JSPS Research Fellow with a research grant (DC1, 21J20658).
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Shirai, Y., Takahashi, M., Ote, M. et al. DIPA-CRISPR gene editing in the yellow fever mosquito Aedes aegypti (Diptera: Culicidae). Appl Entomol Zool 58, 273–278 (2023). https://doi.org/10.1007/s13355-023-00831-y
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DOI: https://doi.org/10.1007/s13355-023-00831-y