Figure 5

Our designed gRNA1-2 remove C9ORF72 repeat expansion and correct C9ORF72 sense RNA foci in primary cortical cultures. (A) The primary cortical neurons cultured from Cas9/ + or Cas9/ + ; C9-Tg mice were infected with control or gRNA1-2 lentiviral particles (Supplementary Fig. 2). The Cas9/ + mouse constitutively expresses Cas9 (PMID: 25,263,330) and the C9ORF72-BAC transgenic mouse (C9-Tg) carries expanded GGGGCC repeats from patient (C9-Tg mouse line 112, PMID: 26,637,796). The editing site fusions (Δ) were detected by primers shown in Fig. 1A and the PCR conditions (low amount of high GC buffer) were shown in Fig. 1D. The amplicons of C9ORF72 last exon only appeared in the neurons derived from mouse carrying C9-Tg (primers shown in Fig. 1A). Mouse Gapdh served as PCR reaction control. (B) The deletion band (Δ) shown in (A) was applied for both forward and reverse Sanger sequencing. The editing site fusions took place upstream of the PAM sites. (C-E) gRNA1-2 significantly reduced the sense RNA foci produced by the C9ORF72 repeats in the primary cortical cultures derived from Cas9/ + ; C9-Tg mice. Control, control lentiviral particles. Both the percentage of cells containing foci (D) and the number of foci per nucleus (E) were significantly reduced in the dual-gRNA treatment groups. In (C), the dotted line circled nuclei of cultured neurons infected with indicated lentiviral particles. In (D and E), the RNA foci counts from three biological replicates. In (D), the values are presented as mean ± SEM (n = 3). In (E), we employed “superplot”⁷⁰ to transparently show the spread in the number of foci per nucleus, and the values are presented as mean ± SD. ***p < 0.001 (t-test, SPSS). In (C), scale bar, 10 μm.