Optimized In Vitro CRISPR/Cas9 Gene Editing Tool in the West Nile Virus Mosquito Vector, Culex quinquefasciatus

SIMPLE SUMMARY: Culex mosquitoes are responsible for many established and emerging mosquito-borne diseases worldwide. These mosquitoes serve as the principal global transmission vector of West Nile virus, which is the leading cause of mosquito-borne disease in humans in the United States. Unfortunately, effective therapeutic drugs and vaccines are still lacking for most mosquito-borne diseases, and current vector controls (e.g., insecticide use) have fallen short of eradicating the disease burden. These concerns highlight the importance of developing novel strategies to prevent arbovirus transmission by Culex mosquitoes, especially as the prevalence of Culex-borne diseases increases globally. Underlying such approaches is often an improved understanding of virus–mosquito interactions. To investigate the putative antiviral role of Culex immune genes, we developed and characterized a Culex-optimized CRISPR/Cas9 plasmid for use in Culex quinquefasciatus-derived (Hsu) cell cultures. We showed that this newly constructed plasmid allows for efficient and reliable codelivery of all CRISPR reagents in vitro in a single plasmid system. These findings suggest that this tool may serve as a valuable resource for the establishment of mutant Hsu-derived cell populations, enabling the identification of mosquito host genes involved in antiviral response. Elucidating gene functions supports the development of alternative gene-based vector control strategies for Culex mosquitoes. ABSTRACT: Culex quinquefasciatus mosquitoes are a globally widespread vector of multiple human and animal pathogens, including West Nile virus, Saint Louis encephalitis virus, and lymphatic filariasis. Since the introduction of West Nile virus to the United States in 1999, a cumulative 52,532 cases have been reported to the CDC, including 25,849 (49.2%) neuroinvasive cases and 2456 (5%) deaths. Viral infections elicit immune responses in their mosquito vectors, including the RNA interference (RNAi) pathway considered to be the cornerstone antiviral response in insects. To investigate mosquito host genes involved in pathogen interactions, CRISPR/Cas9-mediated gene-editing can be used for functional studies of mosquito-derived cell lines. Yet, the tools available for the study of Cx. quinquefasciatus-derived (Hsu) cell lines remain largely underdeveloped compared to other mosquito species. In this study, we constructed and characterized a Culex-optimized CRISPR/Cas9 plasmid for use in Hsu cell cultures. By comparing it to the original Drosophila melanogaster CRISPR/Cas9 plasmid, we showed that the Culex-optimized plasmid demonstrated highly efficient editing of the genomic loci of the RNAi proteins Dicer-2 and PIWI4 in Hsu cells. These new tools support our ability to investigate gene targets involved in mosquito antiviral response, and thus the future development of gene-based vector control strategies.