bioRxiv preprint doi: https://doi.org/10.1101/2021.02.10.430702; this version posted February 11, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. Optimized CRISPR tools and site-directed transgenesis in Culex quinquefasciatus mosquitoes for gene drive development. 1 1 2,3 1 1 Xuechun Feng , Víctor López Del Amo , Enzo Mameli , Megan Lee , Alena L. Bishop , 2,4 1* Norbert Perrimon , Valentino M. Gantz . 1 Section of Cell and Developmental Biology, University of California San Diego, La Jolla, CA 92093. 2 Department of Genetics, Blavatnik Institute, Harvard Medical School, Boston, MA 02115. 3 National Emerging Infectious Diseases Laboratories (NEIDL), Boston University, Boston, MA 02118. 4 HHMI, Harvard Medical School, Boston, MA 02115. *Correspondence:
[email protected] Keywords: population engineering, insect control, mosquito, Culex, CRISPR, HDR, gene drive. ABSTRACT 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.