Fig. 1: Effects of direct repeat lengths on DNA repair outcomes at the kmo locus. | Communications Biology

Fig. 1: Effects of direct repeat lengths on DNA repair outcomes at the kmo locus.

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

Fig. 1

a Schematic representation of pSSA-KmoDR0.2-SceI and pSSA-KmoDR0.7-SceI transgenes integrated into the Ae. aegypti genome resulting in kmoDR0.2-SceI and kmoDR0.7-SceI strains, respectively. Direct repeat (DR) sequences of kmo exons 3/2 (yellow bars) and chromosomal counterparts (pink bars); the I-SceI-induced DSB site at DsRED used to trigger the SSA pathway is indicated, along with locations of PCR primers utilized to confirm insertions (arrows). The DSBs induced at the I-SceI site are processed through NHEJ to disrupt DsRED (Kmo-; DsRED-; EGFP+) or SSA to restore kmo with the deletion of transgenes (Kmo+; DsRED−; EGFP−). b PCR-based verification of insertion site for each transgenic mosquito strain. c The schematic workflow of the SSA test in a split system, where kmoDR0.2-SceI or kmoDR0.7-SceI male mosquitoes were crossed with nuclease-providing females. F1 offspring females were outcrossed with kmo-/- males and individually oviposited in a 24-well tissue culture plate with 2% low melting point agarose. F2 larvae per female were scored for DNA repair event-dependent marker phenotypes. W, white eyes; G, EGFP+ body; R, DsRED+ eyes; Blk, black eyes. d DNA repair events were scored for F1 female founders (triplicate groups). Each bar represents percentage of F1 females that produced at least one NHEJ (WG, green bar) or SSA (Blk, orange bar) progeny. [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 < 0.01; ****P < 0.0001. e The percentage of F2 offspring per individual F1 female for each DNA repair pathway-dependent marker phenotypes: 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 < 0.05; ns not significant.

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