FIG 4.

Lasting interference effect of the CasRx system. (A) Schematic representation of intracranial injection of the CasRx. (B) Expression of CP after RGNNV infection 12 h, 24 h and 48 h at different groups which were injected with CasRx-dNLS and different crRNAs (CP2, RdRp2, or mix). (C) Expression of RdRp after RGNNV infection 12 h, 24 h and 48 h at different groups which were injected with CasRx-dNLS and different crRNAs (CP2, RdRp2, or mix). (D) Expression of CP after RGNNV infection 12 h, 24 h and 48 h at different groups which were injected with CasRx-NLS and different crRNAs (CP2, RdRp2, or mix). (E) Expression of RdRp after RGNNV infection 12 h, 24 h and 48 h at different groups which were injected with CasRx-NLS and different crRNAs (CP2, RdRp2, or mix). (F and G) Brain histology of fish injected with or without the CasRx system. (F) Brain histology at different time points (12 h, 24 h, and 48 h) when injected with CasRx-dNLS and crRNAs targeting the RGNNV CP gene (CP2), RdRp gene (RdRp2), or both (mix). The black arrows indicate vacuoles. Scale bar = 10 μm. (G) Brain histology at different time points (12 h, 24 h, and 48 h) when injected with CasRx-NLS and crRNAs targeting the RGNNV CP gene (CP2), RdRp gene (RdRp2), or both (mix). The black arrows indicate vacuoles. Scale bar = 10 μm. (H) Values indicate the cumulative mortality rate for each group of orange-spotted groupers during the 14-day experimental period after different injection treatments (mock, Lipofectamine 3000, CasRx-dNLS plus ns-crRNA, CasRx-dNLS plus CP2-crRNA, CasRx-dNLS plus RdRp2-crRNA, or CasRx-dNLS plus mix-crRNA). (I) Values indicate the cumulative mortality rate for each group of orange-spotted groupers during the 14-day experimental period after different injection treatments (mock, Lipofectamine 3000, CasRx-NLS plus ns-crRNA, CasRx-NLS plus CP2-crRNA, CasRx-NLS plus RdRp2-crRNA, or CasRx-NLS plus mix-crRNA). ***, P < 0.001; ****, P < 0.0001.