CRISPR/Cas9 gene editing in the West Nile Virus vector, Culex quinquefasciatus Say

Abstract

Culex quinquefasciatus Say is an opportunistic blood feeder with a wide geographic distribution which is also a major vector for a range of diseases of both animals and humans. CRISPR/Cas technologies have been applied to a wide variety of organisms for both applied and basic research purposes. CRISPR/Cas methods open new possibilities for genetic research in non-model organisms of public health importance. In this work we have adapted microinjection techniques commonly used in other mosquito species to Culex quinquefasciatus, and have shown these to be effective at generating homozygous knock-out mutations of a target gene in one generation. This is the first description of the kmo gene and mutant phenotype in this species.

Fig 1. Images of wild-type and kmo knock-out mosquitoes.

Larvae (A), male pupae (B), female pupae (C), male adults (D), and female adults (E). Images were obtained using a Leica MZ165FC microscope with Leica DFC camera (Leica Biosystems, Wetzlar, Germany).

Fig 2. Clustal W alignment of Kmo proteins of A. aegypti and C. quinquefasciatus.

Orthologue alignment generated within Vectorbase for A. aegypti AAEL008879 1-to-1 orthologue in C. quinquefasciatus genome.

Fig 3. Culex quinquefasciatus kmo gene and mutations observed.

Structure of CPIJ017147 as annotated in Vectorbase, sequences of exon 2 through 5 were confirmed in our lab strain and three sgRNA targets in exon 5 were selected (A). White eyed G₁ individuals were collected for genomic DNA extraction, PCR, and sequencing of the kmo locus. All sequenced mutations are presented (B) followed by the size of the indel, and in brackets the number of sequenced alleles with that mutation. All sequenced alleles contained deletions derived from sgRNAs 935 and 936.