Fig. 6: Simultaneous and orthogonal regulated gene knockdown in mice.

A Experimental timeline of in vivo demonstration of GA and ABA-inducible knockdown of Fluc and Antares, respectively by Cas13d and Cas13b. Plasmids carrying the inducible split Cas13 effectors, gRNAs, and the target luciferase are delivered in vivo using hydrodynamic tailed vein transfection at 0 h. Inducers were injected intraperitoneally at 2 h and 6 h after transfection. In vivo luminescence imaging was performed 8 h after transfection for Fluc expression and 24 h after transfection for Antares expression. i.v., intravenous; i.p., intraperitoneal. B The GA-inducible Cas13d generated 98% reduction in Fluc luminescence in the GA-inducible targeted group compared to uninduced group in vivo. Sample replicates left to right: n = 6, n = 3, n = 10, n = 6; P = 0.00641. C The ABA-inducible Cas13b generated 65% reduction in Antares luminescence in the ABA-inducible targeted group compared to uninduced group in vivo. Sample replicates left to right: n = 3, n = 3, n = 4, n = 4; P = 8.56E-5. D Mice were transfected with both Fluc targeting GA-inducible Cas13d system and Antares targeting ABA-inducible Cas13b system, randomly injected with either vehicle control, GA, ABA, or GA and ABA. Luminescence imaging shows that Fluc and Antares were orthogonally regulated by GA and ABA in vivo. Sample replicates left to right: n = 8, n = 5, n = 6, n = 4; P-values from top to bottom: p = 0.0177, p = 0.00788, p = 2.93E-05, p = 0.00397, p = 5.64E-05, p = 0.00504, p(left) = 0.0397, p(right) = 0.0346. P-values were calculated as a 2-tailed t-test. Data are presented as mean values +/− SEM. Error bars indicate the SEM for at least three biological replicates (n ≥ 3). Source data are provided as a Source Data file.