Fig. 2: Site-directed transgenesis of the vasa-Cas9 cassette.

a Three constructs generated for transgenesis in Culex quinquefasciatus. Pink shading highlights the location of the homology arms used for site-specific targeting of the Cq-vasa > Cas9 and Opie2 > DsRed transgenes. An additional Cq-U6:1 > cd1-gRNA transgene is present beyond the right homology arm, preventing its insertion. The representation of the genetic elements is not to scale. b Overview of the gRNA scaffold variants used in this study compared to the native fold of the crRNA/tracrRNA pair. The gray shaded area in the figure highlights the synthetic portions of the gRNA variants that were introduced to link the crRNA and the tracrRNA. Red indicated the synthetic loop. Purple indicates the mutation introduced in the gRNA scaffold. c Injection and cross scheme used to simultaneously evaluate cutting and transgenesis efficiency of the injected plasmids. d Bar graph representing the cutting and transgenesis rates observed in our experimental conditions. The fraction of germlines showing editing (black) or transgenesis (red) over the total germline sampled (n) is reported below each condition. O: “Original” gRNA, L: “Loop”, L + M: “Loop + Mutation”.