Fig. 2. In vitro gene editing of embryonic primary cortical neurons results in a decrease of pathological hallmarks in C9ORF72 ALS/FTD.
a Schematic of harvest and AAV9-delivered Cas9 and gRNA treatment of different C9ORF72 BAC-transgenic mouse cortical primary neurons. Homozygous C9ORF72 BAC-transgenic mice were crossed with WT mice, resulting in heterozygous mice. To generate BAC111/Cas9 mice, homozygous BAC111 mice were crossed with homozygous B6J.129(Cg)-Gt(Rosa)26Sortm1.1(CAG-cas9*,-EGFP)Fezh/J resulting in heterozygosity of both BAC111 C9ORF72 and Cas9. Cells were harvested at embryonic day (E) 15 and transduced with AAV9-gRNA2,3, AAV9-gRNA2,4, and controls at the day in vitro (DIV) 4 and collected at DIV10 for further analyses. BAC111 and C9-500 primary neurons were treated with both AAV9–SpCas9 and AAV9-gRNA2,3 or AAV9-gRNA2,4 and BAC111/Cas9 with only AAV9-gRNA2,3 or AAV9-gRNA2,4. b Genomic DNA of treated BAC111/Cas9, BAC111, and C9-500 primary neurons was amplified with primers flanking gRNA seed sequences, and PCR products were resolved by electrophoresis. The expected band for gRNA2,3 and gRNA2,4-edited DNA is 680 bps. No band is expected for unedited DNA. c Relative expression of poly-GP in BAC111 and C9-500 primary neurons treated with AAV9-Cas9, and AAV9-gRNA2,3 or AAV9-gRNA2,4, assayed by sandwich immunoassay. Data represent mean ± SEM, n = 3 per experimental group, one-way ANOVA, Dunnett, **P = 0.0093 and 0.0016, ***P = 0.0007 and 0.0007. d Representative images of sense RNA foci visualized by fluorescent in situ hybridization (FISH) in BAC111/Cas9 and C9-500 primary neurons treated with AAV9-gRNA2,3 and AAV9-gRNA2,4 for 6 days. Scale bars represent 10 μm. e Quantification of sense foci in treated and untreated BAC111/Cas9 and C9-500 primary neurons. The bar graph represents average percent of nuclei containing 0, 1–5, 6–10, and 11+ foci. Three independent experiments and >600 nuclei counts per sample. Two-way ANOVA, Tukey: **P = 0.0018, ****P < 0.0001. f ddPCR analysis of mRNA expression levels of variants V1, V3, and all variants jointly (V-all) after AAV9-gRNA2,3 and AAV9-gRNA2,4 treatment in C9-500 primary neurons. Expression levels were normalized to TBP and compared to the PBS-treated control. Mean ± SEM, PBS n = 8, treated groups n = 9, one-way ANOVA, Dunnett, *P = 0.0323 and 0.0238, **P = 0.0044. g Densitometric quantification of the C9ORF72 long isoform in C9-500 primary neurons treated with AAV9-gRNA2,3 and AAV9-gRNA2,4. C9 protein levels were normalized against total protein levels. Levels from primary neurons from wild-type mice were averaged and subtracted as a background signal. Mean ± SEM, n = 3, one-way ANOVA, Dunnett, differences between PBS and AAV9-gRNA2,3, AAV9-gRNA2,4-treated cells were not significant.