Fig. 5: Germline expressed Y2-I-AniI predominately triggers NHEJ, and not SSA, at the kmo locus. | Communications Biology

Fig. 5: Germline expressed Y2-I-AniI predominately triggers NHEJ, and not SSA, at the kmo locus.

From: Transgene removal using an in cis programmed homing endonuclease via single-strand annealing in the mosquito Aedes aegypti

Fig. 5

a Schematic representation of pSSA-KmoDR0.7-SceI and pSSA-KmoDR1.5-AniI integrated into genomes of kmoDR0.7-SceI and kmoDR1.5-SceI strains, respectively. The I-SceI or Y2-I-AniI target site was engineered at DsRED to trigger the SSA pathway. b PCR-based verification of transgenic mosquitoes. The PCR primer pair of KmoEx4-F and DsRED-5Ra was used to confirm the identity of the nuclease site engineered in each strain (Bold); The underlined sequence designates the overhang resulting from nuclease cleavage. c Each bar represents the proportion of F1 females that produced at least one NHEJ (WG, green bar) or SSA (Blk, orange bar) progeny across three independent experiments. [F1 total (n)], the total number of F1 female founders; [marker (n or %)], the number or percentage of all F1 females that produced at least one NHEJ or SSA progeny; [SSA/NHEJ], the ratio of [marker (%)] (note that founders that produced both types of events would be counted in both). Tukey’s multiple comparisons test (1-way ANOVA): *P < 0.05; ****P < 0.0001; ns not significant. d Proportion of F2 offspring exhibiting either NHEJ or SSA phenotypes per individual F1 female: WG for NHEJ [% WG/(WGR+WG+Blk)]; Blk for SSA [% Blk/(WGR+WG+Blk)]. Each dot represents rates of NHEJ and SSA events recovered in the progeny of a single F1 female. [F2 WGR (n)], the total number of F2 WGR mosquitoes; [marker (n or %)], the number or percentage of F2 progenies that have the NHEJ (green) or SSA (orange) phenotype; [SSA/NHEJ], the proportional ratio of [marker (%)]. Tukey’s multiple comparisons test (1-way ANOVA): ***P < 0.0001; ns not significant.

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