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

Abstract

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.

CRISPR-based gene drives generate super-Mendelian inheritance in the disease vector Culex quinquefasciatus.

“…If a successful homing event occurred, it would be identi ed by the linkage of the white-gRNA6 element with this unique marker mutation on the receiver "m" chromosome. This marker mutation, hereon referred to as w4-, was repurposed from a white mutant line that we previously built 27 , which carries a disruption of the white coding sequence under the action the white-gRNA4, targeting exon 3 (w4 mutation, Fig. 1c, Fig.…”

“…The Culex vasa-Cas9 homozygous line used in the split-drive was a HDR-based transgenic line generated previously 26 . The w4-/w4mutant line used for tracking conversion rates in a marked chromosome gene drive was built previously and maintained in the lab for several generations 27 . Mosquitoes were reared at 27±1°C, 75% humidity, and a 12h light/dark cycle in the insectary room at the University of California, San Diego.…”

“…quinquefasciatus genome [26][27][28][29] , we set out to assess whether the mechanism underlying homing drives could be demonstrated in this important pest species. Using a 'split-drive' design with our previously developed vasa-Cas9 source integrated at the cardinal locus 26 (a separate, non-homing locus) and gRNAs ('homing elements') integrated into either the white or kynurenine 3-monooxygenase (kmo) loci (both genes involved in eye pigment synthesis pathways and thus providing a readily scorable knockout phenotype 27,29 ) we observed signi cant homing at both locations. The success of this initial proof-of-principle test, at multiple loci, is promising for the further development of gene drives for population manipulation in Culex mosquitoes.…”

“…Having recently developed CRISPR tools necessary for site-speci c integration of transgenes into the Cx. quinquefasciatus genome [26][27][28][29] , we set out to assess whether the mechanism underlying homing drives could be demonstrated in this important pest species. Using a 'split-drive' design with our previously developed vasa-Cas9 source integrated at the cardinal locus 26 (a separate, non-homing locus) and gRNAs ('homing elements') integrated into either the white or kynurenine 3-monooxygenase (kmo) loci (both genes involved in eye pigment synthesis pathways and thus providing a readily scorable knockout phenotype 27,29 ) we observed signi cant homing at both locations.…”

CRISPR-based gene drives generate super-Mendelian inheritance in the disease vectorCulex quinquefasciatus

“…Having recently developed CRISPR tools necessary for site-specific integration of transgenes into the Cx. quinquefasciatus genome [26][27][28][29] , we set out to assess whether the mechanism underlying homing drives could be demonstrated in this important pest species. Using a 'split-drive' design with our previously developed vasa-Cas9 source integrated at the cardinal locus 26 (a separate, non-homing locus) and gRNAs ('homing elements') integrated into either the white or kynurenine 3-monooxygenase (kmo) loci (both genes involved in eye pigment synthesis pathways and thus providing a readily scorable knockout phenotype 27,29 ) we observed significant homing at both locations.…”