Fig. 1

Generation and validation of HttKO clones in SH-SY5Y cells. A A diagram depicting the exons of human Htt and four gRNA sequences. The structural map of Htt is generated from the UCSC Genome Browser. Exon 8 is enlarged in the box with the coded amino acid sequence at the bottom. Red sequence indicates the gRNA2 sequence. B Estimation of mutation frequencies by mismatch cleavage assay. Forty-eight hours after electroporation, genomic DNA from different gRNA- transfected cells was extracted and the fragment around the gRNA targeted sequence was amplified by PCR, cleaved by mismatch assay and analyzed by DNA gel electrophoresis. Upper panel: PCR products before re-annealing and cleavage. Lower panel: PCR products after mismatch cleavage assay. C Sanger sequencing of WT, B7 and C12 clones to identify InDels. D Representative confocal images showing the absence of Htt expression in B7 and C12 clones. All images were taken with the same laser intensity and gain. Not further adjustments were applied. Cells were immunostained with rabbit monoclonal Htt antibodies (D7F7). Nuclei were counterstained with DAPI. Scale bar: 20 μm. E Western blot analysis of Htt expression in WT SH-SY5Y cells, B7 and C12 HttKO clones. Rabbit monoclonal Htt antibody (D7F7) and mouse monoclonal β-actin antibody were used in the blot