Silencing of end-joining repair for efficient site-specific gene insertion after TALEN/CRISPR mutagenesis inAedes aegypti

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.

Highly efficient Cas9-mediated gene drive for population modification of the malaria vector mosquitoAnopheles stephensi

“…stephensi (15) was injected with a solution containing 100 ng/μL each of the pAsMCRkh2 plasmid, Cas9 protein, Cas9 doublestranded RNAs (dsRNAs), and Ku70 dsRNA. The rationale for including the dsRNAs was to silence expression of the incoming Cas9 gene (dsCas9) carried on the plasmid and to reduce activity of the nonhomologous end-joining (NHEJ) pathway (dsKU70) (31) to favor HDR-mediated insertion of the pAsMCRkh2 cargo. Genomic integration of the transgene was achieved by the coinjected Cas9 protein together with the kh2 gRNA encoded on the (28) and square (29).…”

Regulation of physiological processes by microRNAs in insects

“…However, the recent application of Gal4-UAS systems with the miR-SP transgenic method has begun to shed new light on understanding miRNA function in non-drosophilid insects [19, 41]. Additionally, investigations of the CRISPR-Cas9 system offers promising results that will allow genome engineering previously not feasible for non-model organisms [58–62]. …”

Delivery strategies of the CRISPR-Cas9 gene-editing system for therapeutic applications

“…For example, Mashiko demonstrated that microinjection of a plasmid encoding Cas9 and sgRNA into the pronucleus of mouse zygotes is a simple and convenient method to obtain a knockout mouse model within a month 52,53 . Similarly, microinjection of CRISPR-Cas9 was used to edit genes in the cells of rabbits 54 , zebrafish 55 , Ciona intestinalis 56 , worms 57 and Aedes aegypti 58 .…”