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.

Ommochrome pathway genes kynurenine 3-hydroxylase and cardinal participate in eye pigmentation in Plutella xylostella

“…It has been reported in B. mori that the disruption of kmo (w-1 strain) causes a white-eye phenotype, consistent with the mutant phenotype of other ommochrome pathway genes scarlet (w-2 line) and white (w-3 line) [18,19]. Similar white-eye phenotypes were also reported in kmo-deficient mutants of T. castaneum and mosquitoes [12,20,24]. It is noted that the above mutants in B. mori were also accompanied with color changes in egg shell.…”

“…Considering the ease with which they can be visually screened, eye pigmentation genes have become useful tools for identifying germline transformation and gene editing events in several insect species. Here, we identified the P. xylostella homologs of kmo and cardinal , which have been reported as important genes involved in ommochrome synthesis of mosquitoes and silkworm [ 13 , 20 , 24 ] and characterized their mutant phenotypes using the CRISPR/Cas9 system. Relatively high rates of somatic editing (≥ 71.3% in Pxkmo and ≥ 56.4% in Pxcardinal ) and germline transformation (≥ 17.0% in Pxkmo and ≥ 57.1% in Pxcardinal ) were achieved, compared with our previous reports targeting different genes in P. xylostella [ 25 , 27 ].…”

“…The synthesis of all sgRNAs was carried out as described previously [24]. In brief, DNA templates for in vitro synthesis of sgRNAs, which targeted Pxkmo (i.e.…”

“…Genes involved in ommochrome pathway are represented in italics, while the kmo and cardinal genes investigated in this study are indicated with dashed boxes. The color of the pigments/precursors was highlighted with yellow, red or brown boxes drosophilid species [13,19,23,24], the biological functions of these two genes remain unexplored in DBM. In the current study, the CRSPR/Cas9 system, which has been previously demonstrated in DBM [25], was utilized to generate Pxkmo and Pxcardinal mutant moths.…”

“…Despite the essential roles of ommochrome pathway genes kmo and cardinal in eye pigmentation of some non-drosophilid species [ 13 , 19 , 23 , 24 ], the biological functions of these two genes remain unexplored in DBM. In the current study, the CRSPR/Cas9 system, which has been previously demonstrated in DBM [ 25 ], was utilized to generate Pxkmo and Pxcardinal mutant moths.…”