Abstract
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.
Highlights
Conventional control strategies for mosquito-borne pathogens such as malaria and dengue are being complemented by the development of transgenic mosquito strains reprogrammed to generate beneficial phenotypes such as conditional sterility or pathogen resistance
Unlike homing endonucleases, which are difficult to re-engineer, site-specific nucleases based on the type II clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 are unrivaled in the ease with which they can be tailored to a wide range of potential target sequences, already leading to the proposal of alternative gene drive designs and regulatory structure based on these more facile genetic tools [5, 6]
transcription activator-like effector nucleases (TALENs)-mediated gene editing has been demonstrated in mosquitoes [9, 10], the influence of different TALEN architectures on the induction of DSBs has not been examined
Summary
Conventional control strategies for mosquito-borne pathogens such as malaria and dengue are being complemented by the development of transgenic mosquito strains reprogrammed to generate beneficial phenotypes such as conditional sterility or pathogen resistance. 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. We developed and validated a two-step process for performing high-efficiency site-specific insertion of genetic material into the mosquito genome by first evaluating candidate site-specific nucleases in a rapid format, followed by germ line-based editing where the choice of DNA repair response is constrained This model should significantly accelerate both basic and applied research concerning disease vector mosquitoes
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