Fig 1.
Development of CRISPR reagents targeting Ae. aegypti genes involved in flight.
(A) Workflow for performing embryo assays. Gene models and HRMA analysis following embryonic microinjection of CRISPR/Cas9 reagents with each group of sgRNAs for AeAct-4 (B), myo-fem (C), and Aeflightin (D). For (B-D), boxes represent exons, while cross-hatched areas represent the ORF of the corresponding mRNA. For each, red triangles indicate the locations of sgRNAs that were found to successfully cleave the DNA during embryo assay, and the blue and red boxes under the gene models indicate the approximate HRMA amplicon sizes. Melt curves are displayed for clusters of sgRNAs (indicated as “site” or “exon”), with sgRNA-injected (red) and non-injected (gray) samples.
Fig 2.
Establishment and maintenance of heritable loss-of-function mutations in Ae. aegypti flight genes.
(A) For each gene, the wild type (WT) and mutant sequence is shown. The PAM sites for each sgRNA used in the injection mix for the specified location are highlighted in red. Individuals containing each deletion were outcrossed through the indicated generation, at which point individuals heterozygous for the AeAct-4, myo-fem (B), and Aeflightin (C) mutations were intercrossed. For each cross, the ratio of each potential genotype expected is noted in parentheses, with the individuals with an expected flightless phenotype highlighted in red. For Aeflightin, phenotypic identification of all white-eyed pupae enables their removal before further phenotypic analysis based on flight (see Fig 4) is performed.
Fig 3.
Loss of AeAct-4, myo-fem, or Aeflightin results in flightlessness.
(A) Blinded workflow used to score flight ability without experimenter knowledge of genotype, with subsequent genotyping assays. Photographs of wild type (B), AeAct-4 Δ10/Δ10 (C), myo-fem Δ11/Δ11 (D), or AeflightinΔ4/Δ5 (E) females when resting. The percentage of flightless male (black bars) or female (grey bars) mosquitoes for AeAct-4 (F), myo-fem (G), and Aeflightin (H) based on each genotype. The number above each bar represents the number of individuals displaying the flightless phenotype and were confirmed for the specified genotype via sequencing. The dotted red line indicates 100% flightless.
Fig 4.
Mating competitiveness of male Ae. aegypti deficient in AeAct-4 or myo-fem.
(A) Workflow for performing larval progeny assays. The observed (Obs.) vs. expected (Exp.) number of matings for AeAct-4 (B) and myo-fem (C) males based on the sequenced genotypes of pooled larval progeny obtained from the male mating competitiveness assays. Wild type is defined as +/+ and homozygous mutants are defined as Δ10/Δ10 (for AeAct-4) or Δ11/Δ11 (for myo-fem). Each data point represents one replicate, bar height represents the mean of all replicates, and the error bars indicate standard deviation. Chi square was performed for statistical analysis.
Fig 5.
Neighbor-joining tree of AeAct-4 and related homologs in mosquitoes.
A phylogenetic analysis of AeAct-4 (in bold face) and all paralogs with ≥80% amino acid similarity in mosquitoes and Drosophila. The gene identifiers include Ae. aegypti (AAEL in red), Ae. albopictus (AALF in orange), An. gambiae (AGAP in green), An. darlingi (ADAC in blue), Cu. quinquefasciatus (CPIJ in yellow), and D. melanogaster (FBpp in purple). Female-specific genes are represented in the green shaded area, and male-specific genes are in the blue shaded area. All branch points with >50% support based on 1,000 bootstrap replicates are indicated.
Fig 6.
An M-locus-linked sex distorter gene drive targeting female-specific flight genes.
Male mosquitoes modified to contain a site specific nuclease targeting a haploinsufficient female flight gene (myo-fem) in the M-locus would be released to mate with wild type females. All male progeny from these matings would inherit the nuclease, which would inactivate the intact female flight gene inherited from the mother. All female progeny from these matings would inherit one disrupted allele of the female-specific flight gene and therefore be unable to fly, blood feed, or mate to produce future offspring.