Culex quinquefasciatus is a 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. Clustered regularly interspaced short palindrome repeats‐associated protein 9 (CRISPR‐Cas9) based technologies are a powerful tool for genome engineering and functional genetics and consequently have transformed genetic studies in many organisms. Our objective was to improve upon the limited technologies available for genome editing in C. quinquefasciatus to create a reproducible and straightforward method for CRISPR‐Cas9‐targeted mutagenesis in this vector. Here we describe methods to achieve high embryo survival and mutagenesis rates and we provide details on the injection supplies and procedures, embryo handling and guide RNA (gRNA) target designs. Through these efforts, we achieved embryo survival rates and germline mutagenesis rates that greatly exceed previously reported rates in this vector. This work is also the first to characterize the white gene marker in this species, which is a valuable phenotypic marker for future transgenesis or mutagenesis of this vector. Overall, these tools provide the framework for future functional genetic 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.

The ELAV/Hu family is a conserved multigene family of pan‐neuronal RNA‐binding protein involved in post‐transcriptional regulation in metazoans. In Drosophila, three members of this family, ELAV, RBP9 and FNE, are involved in neuronal differentiation, gene expression regulation and so on. This family is less well characterized in Bombyx mori. Two orthologs BmELAV‐like‐1 (BmEL‐1) and BmELAV‐like‐2 (BmEL‐2) share 55%–71% and 47%–62% identity with that of in Drosophila and humans, respectively. Bmel‐1 is ubiquitously expressed while Bmel‐2 is expressed in the head and ovaries specifically. Proteins encoded by both genes are localized in nuclear and cytoplasm. The weight of body, cocoon, pupae and cocoon shell are differently affected in Bmel‐1−/−2− mutants created using CRISPR/Cas9 technology. Mutations of both genes increase the expression of four silk protein genes, Fib‐L, Fib‐H, P25 and Ser‐1. In addition, the oviposition ability of Bmel‐2− females is decreased. This study not only provides valuable insights into the functional roles of Bmelav‐like genes in the growth, cocoon characters and regulation of silk protein genes expression, but also provides useful information for silkworm variety breeding.