Conventional control strategies for mosquito-borne pathogens such as malaria and dengue are now being complemented by the development of transgenic mosquito strains reprogrammed to generate beneficial phenotypes such as conditional sterility or pathogen resistance. The widespread success of site-specific nucleases such as transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 in model organisms also suggests that reprogrammable gene drive systems based on these nucleases may be capable of spreading such beneficial phenotypes in wild mosquito populations. Using the mosquito Aedes aegypti, we determined that mutations in the FokI domain used in TALENs to generate obligate heterodimeric complexes substantially and significantly reduce gene editing rates. We found that CRISPR/Cas9-based editing in the mosquito Ae. aegypti is also highly variable, with the majority of guide RNAs unable to generate detectable editing. By first evaluating candidate guide RNAs using a transient embryo assay, we were able to rapidly identify highly effective guide RNAs; focusing germ line-based experiments only on this cohort resulted in consistently high editing rates of 24-90%. Microinjection of double-stranded RNAs targeting ku70 or lig4, both essential components of the end-joining response, increased recombination-based repair in early embryos as determined by plasmid-based reporters. RNAi-based suppression of Ku70 concurrent with embryonic microinjection of site-specific nucleases yielded consistent gene insertion frequencies of 2-3%, similar to traditional transposon-or ΦC31-based integration methods but without the requirement for an initial docking step. These studies should greatly accelerate investigations into mosquito biology, streamline development of transgenic strains for field releases, and simplify the evaluation of novel Cas9-based gene drive systems.

A dominant male-determining locus (M-locus) establishes the male sex (M/m) in the yellow fever mosquito, Aedes aegypti. Nix, a gene in the M-locus, was shown to be a male-determining factor (M factor) as somatic knockout of Nix led to feminized males (M/m) while transient expression of Nix resulted in partially masculinized females (m/m), with male reproductive organs but retained female antennae. It was not clear whether any of the other 29 genes in the 1.3-Mb M-locus are also needed for complete sex-conversion. Here, we report the generation of multiple transgenic lines that express Nix under the control of its own promoter. Genetic and molecular analyses of these lines provided insights unattainable from previous transient experiments. We show that the Nix transgene alone, in the absence of the M-locus, was sufficient to convert females into males with all male-specific sexually dimorphic features and male-like gene expression. The converted m/m males are flightless, unable to perform the nuptial flight required for mating. However, they were able to father sex-converted progeny when presented with cold-anesthetized wild-type females. We show that myo-sex, a myosin heavy-chain gene also in the M-locus, was required for male flight as knockout of myo-sex rendered wild-type males flightless. We also show that Nix-mediated female-to-male conversion was 100% penetrant and stable over many generations. Therefore, Nix has great potential for developing mosquito control strategies to reduce vector populations by female-to-male sex conversion, or to aid in a sterile insect technique that requires releasing only non-biting males.

Mosquitoes are high-impact disease vectors with the capacity to transmit pathogenic agents that cause diseases such as malaria, yellow fever, chikungunya, and dengue. Continued growth in knowledge of genetic, molecular, and physiological pathways in mosquitoes allows for the development of novel control methods and for the continued optimization of existing ones. The emergence of site-specific nucleases as genomic engineering tools promises to expedite research of crucial biological pathways in these disease vectors. The utilization of these nucleases in a more precise and efficient manner is dependent upon knowledge and manipulation of the DNA repair pathways utilized by the mosquito. While progress has been made in deciphering DNA repair pathways in some model systems, research into the nature of the hierarchy of mosquito DNA repair pathways, as well as in mechanistic differences that may exist, is needed. In this review, we will describe progress in the use of site-specific nucleases in mosquitoes, along with the hierarchy of DNA repair in the context of mosquito chromosomal organization and structure, and how this knowledge may be manipulated to achieve precise chromosomal engineering in mosquitoes.

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