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. 2021 Sep 9;95(19):e00461-21. doi: 10.1128/JVI.00461-21

FIG 3.

FIG 3

In vivo RGNNV targeting via the CasRx system. (A) Schematic representation of intracranial injection of CasRx. (B) Diagram of intracranial injection location. a, the dotted red line indicates the site of the intracranial injection; b, the dotted red line indicates brain tissue. The red arrow indicates the injection area. (C) GFP expression in the brain of representative fish after injection with Lipofectamine 3000, CasRx-dNLS, or CasRx-NLS. (D and E) Western blot analysis of GFP expression in the brain after plasmid injection. The mouse anti-GFP antibody was used to detect the expression of the CasRx-dNLS (D) and CasRx-NLS (E) systems in the brain. (F to I) Intraperitoneal injection of CasRx prevented RGNNV infection. (F) Expression of CP after injection with CasRx-dNLS and different crRNAs (CP2, RdRp2, or mix). (G) Expression of RdRp after injection with CasRx-dNLS and different crRNAs (CP2, RdRp2, or mix). (H) Expression of CP after injection with CasRx-NLS and different crRNAs (CP2, RdRp2, or mix). (I) Expression of RdRp after injection with CasRx-NLS and different crRNAs (CP2, RdRp2, or mix). (J) Representative RGNNV FISH staining of brain sections from fish injected with the CasRx system (n = 3). Little fluorescence was observed after injection with CasRx-dNLS or CasRx-NLS targeting CP2-crRNA, RdRp2-crRNA, or mix-crRNA. Scale bar = 50 μm. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.