Fig. 2. Identification of a functional cis-acting PD-associated SNP in an intronic enhancer element of SNCA.

(a) Heatmap of H3K4me1 and H3K27ac ChIP-Seq and DHSs-enrichment tracks for several CNS regions in the SNCA locus (for details see Extended Data Fig. 3a; Data provided by NIH Roadmap Epigenomics Consortium; http://www.roadmapepigenomics.org). Shown are the locations of NACP-Rep1 and PD-associated SNPs overlapping with two proximal enhancer elements (3′UTR-enhancer and intron-4-enhancer) highlighted by light gray boxes. (b) Schematic illustration of the CRISPR/Cas9-mediated strategy to delete and subsequently insert intron-4 enhancer elements with indicated PD-associated risk SNP genotype at rs356168 and rs3756054. Targeted clones carrying inserted risk alleles in cis with the A-(FAM)-reporter SNP (confirmed by genomic sequencing-based phase-reconstruction) were used for subsequent analysis described in (c,d). (c, d) Relative allele-specific SNCA expression in (c) neural precursors and (d) mixed neuronal cultures (differentiation day 25) derived from targeted cell lines with indicated intron-4 enhancer alleles compared to hESCs carrying homozygous enhancer deletions (ΔE4/ΔE4) (expression was normalized relative to ΔE/ΔE cell lines). Data are presented as dot plot; each dot represents mean of 3 technical replicates. Allele-specific expression for each clone was analyzed in 3 independent biological replicate experiments and combined according to genotypes. Black lines indicate mean expression for each genotype; n indicates number of independently targeted clones per genotype, † indicates an additional sub-clone derived from one of the two targeted clones for this genotype. Statistical differences between genotypes were calculated using one-way ANOVA (alpha = 0.05) followed by Tukey’s multiple comparison test based on allele-specific expression of all biological replicates. *P < 0.001; ** P<0.0001; source data and detailed statistical analysis are provided as Source Data for Extended Data Figure 2.