Figure 2.

AAV delivery of the CRISPR-hfCas13x system restores paternal Ube3a expression in AS mouse brain

(A) TdTomato signal in brain and liver of WT mouse at 3 weeks after I.C.V. injection of 1.9 × 10¹¹ or I.V. injection of 0.3 × 10¹¹ AAV-PHP.eb particles containing CAG-tdTomato at P0. Scale bar, 500 μm. (B) Experimental timeline for evaluation in WT and AS (Ube3a^m−/p+) mice after I.C.V. injection of AAV-PHP.eb containing hSyn1-hfCas13x.1/U6-NT or hSyn1-hfCas13x.1/U6-cr9 at P0. (Cand D) RT-qPCR analysis of mRNA expression of Ube3a and Ube3a-ATS in the cerebral cortex (Cor.) (C) and hippocampus (Hip.) (D) of WT and AS mice with indicated treatment at 4 weeks (n = 3 for all groups). (E and F) Western blot (E) and quantification (F) of protein expression in the cerebral cortex (cor.) and hippocampus (hip.) of WT and AS mice with indicated treatment at 4 weeks (n = 3 for all groups). (G and H) Immunofluorescence staining for UBE3A with FLAG in cortex (G) or hippocampus (H) of WT or AS mice with indicated treatment at 4 weeks. Scale bar, 100 μm. Statistical significance was assessed by one-way ANOVA followed by Tukey’s multiple comparison test. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. ns, not significant.