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. 2023 Feb 18;31(7):2286–2295. doi: 10.1016/j.ymthe.2023.02.015

Figure 1.

Figure 1

Screen of crRNAs targeting the lncRNA Ube3a-ATS to unsilence paternal Ube3a

(A) Schematic diagram of the Ube3a locus in the mouse genome. IC, imprinting center. (B) Ube3a-ATS mRNA expression levels under targeting by different crRNAs in N2a cells (n = 2 for all groups). (C) RT-qPCR analysis of mRNA expression in wild-type (WT) primary cultured neurons of C57 mice. The WT neurons were infected with lentivirus containing EFS-hfCas13x.1/U6-cr8 (WT + cr8), EFS-hfCas13x.1/U6-cr9 (WT + cr9), or the non-targeted control crRNA EFS-hfCas13x.1/U6-NT (WT + NT) (n = 3 for all groups). (D and E) Western blot analysis (D) and quantification of band density (E) of UBE3A protein expression in cultured primary neurons of WT or AS mice infected with lentivirus-containing hSyn1-hfCas13x.1/U6-NT or hSyn1-hfCas13x.1/U6-cr9 (n = 3 for all groups). (F) RT-qPCR analysis of Ube3a mRNA levels in cultured primary neurons of WT or AS mice infected with hSyn1-hfCas13x.1/U6-NT or hSyn1-hfCas13x.1/U6-cr9 (n = 3 for all groups). (G) Expression of the indicated genes in cultured primary neurons of AS mice infected with hSyn1-hfCas13x.1/U6-cr9 to silence the paternal lncRNA Ube3a-ATS (n = 3 for all groups). Levels were relative to that in primary neurons of AS mice infected with the hSyn1-hfCas13x.1/U6-NT non-targeted control. 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.