CRISPR/Cas-9 mediated knock-in by homology dependent repair in the West Nile Virus vector Culex quinquefasciatus Say

Abstract

Culex quinquefasciatus Say is a mosquito distributed in both tropical and subtropical regions of the world. It is a night-active, opportunistic blood-feeder and vectors many animal and human diseases, including West Nile Virus and avian malaria. Current vector control methods (e.g. physical/chemical) are increasingly ineffective; use of insecticides also imposes hazards to both human and ecosystem health. Advances in genome editing have allowed the development of genetic insect control methods, which are species-specific and, theoretically, highly effective. CRISPR/Cas9 is a bacteria-derived programmable gene editing tool that is functional in a range of species. We describe the first successful germline gene knock-in by homology dependent repair in C. quinquefasciatus. Using CRISPR/Cas9, we integrated an sgRNA expression cassette and marker gene encoding a fluorescent protein fluorophore (Hr5/IE1-DsRed, Cq7SK-sgRNA) into the kynurenine 3-monooxygenase (kmo) gene. We achieved a minimum transformation rate of 2.8%, similar to rates in other mosquito species. Precise knock-in at the intended locus was confirmed. Insertion homozygotes displayed a white eye phenotype in early-mid larvae and a recessive lethal phenotype by pupation. This work provides an efficient method for engineering C. quinquefasciatus, providing a new tool for developing genetic control tools for this vector.

Mapping and CRISPR homology-directed repair of a recessive white eye mutation in Plodia moths

“…The impact of longer repair template insertion on knock-in efficiency is yet to be determined. Efficient insertion of larger fragments in insects have been reported using donor dsDNA template, including the use of long donor dsDNA plasmids for HDR ( Gilles et al., 2015 ; Ma et al., 2014 ; Purusothaman et al., 2021 ), microhomology-mediated end joining in Bombyx ( Nakade et al., 2014 ), and the homology-independent end-joining of plasmid cassettes ( Bosch et al., 2020 ; Matsuoka et al., 2021 ). These approaches are promising avenues for optimizing larger cassette insertion in Plodia genome, the third lepidopteran organism after Bombyx, and Plutella where germline-heritable CRISPR HDR edits are reported ( Takasu et al., 2016 ; Wang et al., 2020 ).…”

CRISPR/Cas9 mediates efficient site‐specific mutagenesis of the odorant receptor co‐receptor (Orco) in the malaria vector Anopheles sinensis

“…aegypti 10,35 and Cu. quinquefasciatus, 38 but significantly lower than the gene knockout frequency mediated by the NHEJ repair pathway, 10,[34][35][36]40 and lower than the transposon mediated transgenic frequency of An. sinensis.…”

“…Since its discovery, the CRISPR/ Cas9 system has revolutionized genome editing and has been successfully applied to a diverse range of insect species, including members of the Diptera, Lepidoptera, Hymenoptera, Hemiptera, Orthoptera, and Coleoptera. 18,[22][23][24][25][26][27][28][29][30][31][32][33] The CRISPR/Cas9 system has been applied in studies on mosquito species such as Aedes aegypt, 10,34,35 Aedes albopictus, 36 Culex quinquefasciatus, 37,38 Anopheles gambiae, 39 Anopheles albimanus, 40 Anopheles coluzzii, 40 Anopheles funestus, 40 and Anopheles stephensi. 41 These studies showed that CRISPR/Cas9 is a strong tool that could be used to uncover the mechanistic knowledge of myriad mosquito biological processing.…”

A multiplexed, confinable CRISPR/Cas9 gene drive propagates in caged Aedes aegypti populations

“…aegypti fitness effects were not measured (18, 39). In our own recent experience with Culex quinquefasciatus kmo -/- could be generated and maintained as homozygotes, but an insertional mutant expressing a fluorescent protein was homozygous lethal (43, 44). Using these best fit parameter values within the stochastic model shows that experimental results fall within the expected range.…”