Fig. 1: dSpCas9 targeted to Snord115 unsilences patUbe3a in the absence of a chromatin modifying domain.
From: AAV-dCas9 vector unsilences paternal Ube3a in neurons by impeding Ube3a-ATS transcription
a, b Representative images of UBE3A-YFP immunofluorescence in tdTomato positive, transfected Ube3am+/pYFP neurons from dSpCas9 negative control and Spjw33 conditions. Scale bar, 50 µm. c The percentage of transfected Ube3am+/pYFP primary mouse cortical neurons positive for UBE3A–YFP following transfection with each gRNA and SpCas9 variant indicated. n = 4 wells per condition. Error bars indicate standard error of the mean (SEM). Unpaired, two-tailed Student’s t-test comparing Spjw33 condition to non-target gRNA condition, *p < 0.05. d Expression of the indicated genes in Ube3am+/pYFP neurons 7 days after transduction with lentivirus delivering dSpCas9 and gRNA Spjw33 relative to dSpCas9 non-target gRNA control (dashed line). Expression normalized to Eif4a2. n = 2 wells per condition. e Schematic of non-template and template strand orientation relative to Ube3a-ATS transcription. Example palindromic gRNA target protospacer adjacent motifs are underlined. f, g Correlation of the percentage of transfected UBE3A-YFP positive neurons transfected with dSpCas9 and gRNAs targeting the (d) template or (e) non-template strand. The number of Snord115 target sites for each gRNA is shown. n = 8–12 wells per gRNA condition. h, i Correlation of UBE3A-YFP unsilencing when neurons were transfected with (f) dSpCas9 or (g) SpCas9 and sets of palindromic gRNAs that target the non-template versus template strand. n = 8–12. Hypergeometric means test, *p < 0.05.
