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. Author manuscript; available in PMC: 2023 Oct 10.
Published in final edited form as: J Vis Exp. 2021 Feb 5;(168):10.3791/62154. doi: 10.3791/62154

Figure 3: Ubiquitous in vivo exogenous RNA targeting using a three-component CasRx system.

Figure 3:

(A) Schematics of the two-step genetic cross. (B) Total inheritance percentages for all genotypes emerging in the F2 generation. Inheriting all three transgenes (Ubiq-CasRx, Ubiq-Fluc-Ubiq-Rluc, and gRNAFLuc) in F2 progeny resulted in 100% lethality and was significantly lower compared to the Ubiq-dCasRx triple transheterozygotes control group (p = 0.001, t-test). (C) Carrying Ubiq-CasRx/gRNAFluc alone or Ubiq-CasRx and Ubiq-Fluc-Ubiq-Rluc alone did not lead to severe lethality, and inheritance ratios between Ubiq-CasRx and Ubiq-dCasRx transheterozygotes were not significantly different (p = 0.41 and p = 0.51, respectively, t-test). (D) Luciferase ratios normalizing Fluc readings to Rluc readings. Triple transheterozygous flies expressing Ubiq-CasRx, Ubiq-Fluc-Ubiq-Rluc, gRNAFLuc were embryonic lethal, which was represented by a fly with an “X”, and as a result luciferase expression was not measured. Fluc/Rluc ratio of Ubiq-CasRx/+, Ubiq-Fluc-Ubiq-Rluc/TM6, Stb transheterozygotes was significantly lower than that of the other Ubiq-Fluc-Ubiq-Rluc-expressing groups (p = 1.2e-06 or lower, t-test). The results from the gRNAFLuc-only group were significantly lower than that of all other groups (p = 1.2e-06 or lower, t-test).