Silencing of end-joining repair for efficient site-specific gene insertion after TALEN/CRISPR mutagenesis in Aedes 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.

Keywords: Aedes; CRISPR; TALEN; recombination; transgenic.

Fig. 1.

Heterodimeric TALEN activity in mosquito embryos. (A) Representation of the TALEN constructs used to target Ae. aegypti kmo. All possessed the same set of TALE repeats with the exception of the final 1/2 repeat, which either perfect-matched (red) or was mismatched (blue) to the target site (block arrow). Constructs also differed in the FokI domain as indicated. Architecture used in ref. is indicated (#). (B) SSA assay to detect TALEN activity in mosquito embryos. Each point represents a group of ∼100 injected embryos; mean and SD are indicated. Groups were found to be significantly different by ANOVA (P < 0.05), with assignment to groups (a, b) by Dunnett's multiple comparison test. (C) HRMA-based analysis of amplicons obtained from mosquito embryo DNA 24 h following injection with the indicated kmo TALEN pair [MM (mismatched) and PM (perfect match)] or from noninjected controls (CNT).

Fig. 2.

Somatic and germ-line CRISPR/Cas9 editing of Ae. aegypti. White-light photographs of WT eyes (A), G₀ individuals showing somatic CRISPR-editing (B and C), and germ line-based CRISPR-edited in G₁ progeny (D). (E) Analysis of indels in CRISPR-edited progeny. Top line represents WT sequence; subsequent lines show individual mutant sequences. Underlined text indicates the crRNA target sequence including the PAM (highlighted in blue) and cleavage point (red letters and black arrow); the kmo-exon5 TALEN spacer site is shown for comparison (gray). Deleted bases are denoted by dashes and inserted bases are shown in blue. The number of deleted (Δ) or inserted (+) bases and their occurrence are indicated to the right; in-frame mutations are indicated (*) to the left.

Fig. 3.

Suppression of NHEJ components increases recombination activity in mosquito embryos. Representation of the plasmid constructs used to detect SSA (A) or NHEJ (B). Relative light units observed from embryos 48 h after injection with the SSA sensor (C) or NHEJ sensor (D) in the presence of the indicated dsRNA. Each point represents a pool of ∼100 embryos. Data were trimmed by removing highest/lowest points from all samples (experimental and control). For C, statistical differences were determined by ANOVA followed by Dunnett's multiple comparison test; statistically different groups are indicated (*P < 0.05; **P < 0.01). For D, statistical differences were determined with a two-tailed t test (Mann-Whitney); P values are indicated.

Fig. 4.

Site-specific HDR-based gene insertion. (A) Representation of the HDR donor construct used to insert the PUb-EGFP expression cassette into exon5 of dcr2. Block arrows represent dcr2 exons 4–8; blue bars indicate sequences used as left (L) and right (R) homology arms in the circular donor construct. TALEN target site is indicated by red vertical line; PCR primers used to confirm integration into the target site are indicated by red arrowheads. (B) Transgenic larvae following site-specific insertion of the PUb-EGFP cassette. (C) PCR confirmation of HDR-mediated site-specific insertion of the PUb-EGFP transgene.