Figure 3.

Both Cas9.P2^on/off and u^MCas9^on are effective at inducing intestinal stem cell derived tumors. (A) Schematic representation of the timepoints used for experiments. Female flies expressing either Cas9.P2^on or u^MCas9^on were crossed to male flies expressing different gRNAs. The cross was maintained at 18 °C and the progeny were raised under these conditions during their development. After adult progeny emerged (T₀), they were shifted to 29 °C continuously for 20 days to induce the esg^ts expression system, which allows for both Cas9 and gRNAs to be expressed in ISCs/EBs. Since high expression of Cas9.P2 is toxic, we also devised an on/off strategy (Cas9.P2 ^on/off), whereby adult progeny were initially shifted to 29 °C for 10 days (T₁₀), then to 18 °C for 30 days (T₄₀) and finally at 29 °C for one day (T₄₁). (B) Schematic representation of the intestine with different regions highlighted. (C) Confocal images of the R4 regions of the intestine in flies expressing Cas9 and gRNAs against hep, Mad, neur, and Notch. Notice that for Cas9.P2^on/off and u^MCas9^on with gRNA against neur and Notch resulted in massive proliferation of ISCs/EBs. (D) Heatmap showing the average percentage of GFP⁺ ISCs/EBs after using different Cas9 enzymes for perturbations. Scale bar: 30 μm.