TY - JOUR T1 - Evaluation of gene knock-outs by CRISPR as potential targets for the genetic engineering of the mosquito <em>Culex quinquefasciatus</em> JF - bioRxiv DO - 10.1101/2020.10.21.349704 SP - 2020.10.21.349704 AU - Xuechun Feng AU - Lukas Kambic AU - Jared H.K. Nishimoto AU - Floyd A. Reed AU - Jai A. Denton AU - Jolene T. Sutton AU - Valentino M. Gantz Y1 - 2021/01/01 UR - http://biorxiv.org/content/early/2021/04/15/2020.10.21.349704.abstract N2 - Culex quinquefasciatus mosquitoes are a globally widespread vector of several human and animal pathogens. Their biology and behavior allow them to thrive in proximity to urban areas, rendering them a constant public health threat. Their mixed bird/mammal feeding behavior further offers a vehicle for zoonotic pathogens transmission to people, and separately, poses a threat to the conservation of insular birds. The advent of CRISPR has led to the development of novel technologies for the genetic engineering of wild mosquito populations, yet research in Culex quinquefasciatus has been lagging compared to other disease vectors. Here we use this tool to disrupt a set of five pigmentation genes in Culex quinquefasciatus that, when altered, lead to visible, homozygous-viable phenotypes. We further validate this approach in separate laboratories and in two distinct strains of Culex quinquefasciatus that are relevant to potential future public health and bird conservation applications. We generate a double-mutant line, demonstrating the possibility of sequentially combining multiple such mutations in a single individual. Lastly, we target two loci, doublesex in the sex-determination pathway and proboscipedia a hox gene, demonstrating the flexibility of these methods applied to novel targets. Our work provides a platform of seven validated loci that could be used for targeted mutagenesis in Culex quinquefasciatus and the future development of genetic suppression strategies for this species. Furthermore, the mutant lines generated here could have widespread utility to the research community using this model organism, as they could be used as targets for transgene delivery, where a copy of the disrupted gene could be included as an easily-scored transgenesis marker.Competing Interest StatementV.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. 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. J.A.D., L.K., F.A.R., J.T.S., J.H.K.N. declare no competing interests. ER -