Figure 6

Evaluating single construct synthetic lethality within protected genomes. (A) Single vectors containing either one (eve) or two (eve & hid1) sgRNA transgenes (for erecting a single barrier or a double barrier) as well as dCas9-VPR driven by different promoters were integrated into the Drosophila melanogaster genome either at AttP9-A (VK00027) on chromosome 3 or by random P-element integration. The target genetic background is homozygous for eveΔ11 in the case of a single barrier or double-homozygous for eveΔ11;hid1-Δ13 in the case of the double barrier experiments. The Mini-White selective marker (not shown) was used to select T0 transgenics in all cases. T1 transgenic lines were tested for associated lethality by scoring for associated red eye marker presence in F1 progeny in crosses to ‘wild-type’ (w1118) virgin females. Shown are the counts of transgenic/total flies from which we calculated the percentage of synthetic lethality of the transgene. 100% indicates the transgene was not inherited at all, 0% indicates that the transgene was inherited at a ratio consistent with no associated lethality. Single barrier (SB), Double barrier using tandem sgRNAs (DB1), Double barrier using sgRNA with a tRNA link (DB2). Promoters used: phow (-H), pαTubulin-84b-Long (-LT), pαTubulin-84b-Short (-ST), ptwist (-TW). (B) qRT-PCR measuring eve expression in embryos from crosses of two P-element lines to w1118 (wild-type) and eveΔ11. Two biological replicates were measured for each condition (black bars) and the mean is shown in red. Error bars show 95% confidence limits, calculated from three technical replicates (See Supplementary Technical Cross 6).