Optimized CRISPR tools and site-directed transgenesis in Culex quinquefasciatus mosquitoes for gene drive development

Culex mosquitoes are a global vector for multiple human and animal diseases, including West Nile virus, lymphatic filariasis, and avian malaria, posing a constant threat to public health, livestock, companion animals, and endangered birds. While rising insecticide resistance has threatened the control of Culex mosquitoes, advances in CRISPR genome-editing tools have fostered the development of alternative genetic strategies such as gene drive systems to fight disease vectors. However, though gene-drive technology has quickly progressed in other mosquitoes, advances have been lacking in Culex . Here, we developed a Culex- specific Cas9/gRNA expression toolkit and used site-directed homology-based transgenesis to generate and validate a Culex quinquefasciatus Cas9-expressing line. We showed that gRNA scaffold variants improve transgenesis efficiency in both Culex and Drosophila and boost gene-drive performance in the fruit fly. These findings support future technology development to control Culex mosquitoes and provide valuable insight for improving these tools in other species. ### Competing Interest Statement V.M.G. is a founder of and has equity interests in Synbal, Inc. and Agragene, Inc., companies that may potentially benefit from the research results described in this manuscript. V.M.G. also serves on both the company's Scientific Advisory Board and the Board of Directors of Synbal, Inc. The terms of this arrangement have been reviewed and approved by the University of California, San Diego in accordance with its conflict of interest policies. X.F., V.L.D.A., E.M., M.L., A.L.B and N.P. declare no competing interests.