Fig. 2: SSA-based transgene elimination can be catalyzed in cis.

a Schematic representation of pSSA-KmoDR0.7-nos-SceI and pSSA-KmoDR1.5-nos-SceI integrated into genomes of kmo^{DR0.7-nos-SceI} and kmo^{DR1.5-nos-SceI} strains, respectively. The kmo exons 3/2 and exons 3/2-intron 1 sequences (yellow bars) were engineered for DR0.7 and DR1.5 motifs with chromosomal counterparts (pink bars), respectively. I-SceI-target site is shown, with I-SceI coding region under the control of the germline-specific nos gene promoter/3′UTR. b PCR-based verification of transgene insertion to verify insertion site, DR length gap between the two strains (KmoEx4-F + DsRED-5Ra), the presence of nos-I-SceI (NosPro-3Fa + SceI-5Ra), and the chromosomal integration of transgenes (SV40-R + In1-IR2). c The schematic workflow of the 1 piece-SSA test over multi-generations. For each generation, females (n = 20–25) carrying the intact transgene (WGR) as confirmed by HRMA were out-crossed with kmo^-/- males. In the next generation, offspring larvae were scored for marker phenotypes to determine DNA repair events: W, white eyes; G, EGFP⁺ body; R, DsRED⁺ eyes; Blk, black eyes. Tukey’s multiple comparisons test (1-way ANOVA): ns not significant. d Each bar represents percentage of G₁₀ females that produced at least one NHEJ (WG, green bar) or SSA (Blk, orange bar) progeny from three independent experimental groups. [G₁₀ total (n)], the total number of G₁₀ female founders; [marker (n or %)], the number or percentage of all G₁₀ 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): ns not significant. e Percent of G₁₁ offspring per individual G₁₀ female for 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 G₁₀ female. [G₁₁ WGR (n)], the total number of G₁₁ WGR mosquitoes; [marker (n or %)], the number or percentage of G₁₁ 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): ns not significant. f PCR analysis of genomic DNAs from white/EGFP⁺ eyed-mosquitoes using indicated primers from G₁₁ mosquitoes. Identified large deletions are shown.