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. Author manuscript; available in PMC: 2016 Oct 20.
Published in final edited form as: Nature. 2016 Apr 20;533(7601):95–99. doi: 10.1038/nature17939

Extended data Figure 10. NACP-Rep1 repeat length has no cis-acting effect on SNCA expression in hESC derived neurons.

Extended data Figure 10

(a) Schematic illustration of the CRISPR/Cas9-mediated genome editing strategy to generate heterozygous deletions (ΔRep1/wild-type) in WIBR3 and BGO1 hESCs and subsequently insert indicated NACP-Rep1 length variants. Displayed are the genomic organization of the SNCA locus, an enlarged view of the wild-type and NACP-Rep1 deleted (ΔRep1) allele, targeting-targeting sequences (underlined, PAM sequence in red), restriction sites and Southern blot (SB) probe. Shown below is targeting vector (TV) design to insert the respective NACP-Rep1 elements (Rep1-257, Rep1-259, Rep1-261 and Rep1-263) with indicated repeat sequences. Only clones with heterozygous deletion of the repeat element on the same chromosome were identified based on the genotype of two heterozygous SNPs (rs58864428 and rs10030935) upstream and downstream of NACP-Rep1 and selected for subsequent experiments. (b) Representative Southern blot analysis of wild type and targeted WIBR3 hESCs with indicated NACP-Rep1 genotypes (modified alleles highlighted in red; no targeted wild-type allele represents NACP-Rep1-261 element). (c) Table summarizing NACP-Rep1 deletions and insertions in WIBR3 and BGO1 hESCs. (d) Representative fragment length analysis confirms expected NACP-Rep-1 repeat length in targeted cell lines. Red line indicates NACP-Rep1-261 peak at 269 bp. (e, f) Analysis of relative allele-specific SNCA expression in neurons (differentiation day 25) derived from targeted cells lines with indicated NACP-Rep1 alleles compared with untargeted controls in WIBR3 (e) and BGO1 (f) hESCs (expression was normalized relative to wild-type cells). Shown are mean values ± s.d. of three independent biological replicate experiments for each individual clone of the indicated genotype. Differences between individual clones or combined clones by genotypes were not significant based on one-way ANOVA testing for multiple comparisons between groups (alpha = 0.05) and did show a repeat length-dependent linear trend analyzed by linear regression. Source data are provided as Source Data for Extended Data Figure 10.