Methods for the generation of heritable germline mutations in the disease vector Culex quinquefasciatus using CRISPR/Cas9.

Abstract

Culex quinquefasciatus is vector of many diseases that adversely impact human and animal health; however, compared to other mosquito vectors limited genome engineering technologies have been characterized for this vector. CRISPR-Cas9 based technologies are a powerful tool for genome engineering and functional genomics and consequently have transformed genomics studies in many organisms. Our objective was to improve upon the limited technologies available for genome editing in Cx. quinquefasciatus to create a reproducible and straightforward method for CRISPR-Cas9-targeted mutagenesis in this vector. Here we describe methods to both improve embryo survival rates as well as mutagenesis rates by optimizing injection supplies and equipment, embryo injection procedures, embryo handling and gRNA target design. Through these efforts, we achieved embryo survival rates and germline mutagenesis rates that greatly exceed any previously reported rates in this vector. This work was also the first characterize the white gene marker, which is a valuable phenotypic marker for future transgenesis or mutagenesis of this vector. In the end, these tools provide the framework for future functional genomic studies in this important disease vector and may support the development of future gene drive and genetic technologies that can be used to control this vector.