Evaluation of Gene Knockouts by CRISPR as Potential Targets for the Genetic Engineering of the Mosquito <i>Culex quinquefasciatus</i>

Abstract

<i>Culex quinquefasciatus</i> 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 into <i>Cx. quinquefasciatus</i> has been lagging compared to other disease vectors. Here, we use this tool to disrupt a set of five pigmentation genes in <i>Cx. quinquefasciatus</i> that, when altered, lead to visible, homozygous-viable phenotypes. We further validate this approach in separate laboratories and in two distinct strains of <i>Cx. quinquefasciatus</i> 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, <i>doublesex</i> in the sex-determination pathway and <i>proboscipedia</i>, 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 <i>Cx. quinquefasciatus</i> 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.