CRISPR knock-out of alpha-synuclein in patient-derived pluripotent stem cell model of Parkinson’s disease

The accumulation and aggregation alpha-synuclein protein (a-syn) is a critical event in Parkinson’s disease (PD) pathophysiology, impairing neuronal function and contributing to dopaminergic neuronal cell death. The pathogenic genomic triplication of the alpha-synuclein (SNCA) gene (chromosomal locus 4q21, size 1.7Mb) in patients results in early onset rapidly progressive parkinsonism with diffuse Lewy body pathology and severe autonomic involvement (1), suggesting a direct link between increased gene expression of wild-type a-syn and disease development. We have previously shown that overexpression of a-syn as it related to the SNCA genomic triplication is linked to increased susceptibility for oxidative stress and impairment of neuronal maturation in patient-derived fibroblasts or induced pluripotent stem cell (iPSC) models (2,3,4). The goal of this study was to combine iPSC technology with gene editing to establish isogenic cellular tools which express varying wild-type SNCA gene copy numbers. With this set of new cell lines, we are able to address what are the physiological and detrimental effects of varying a-syn levels, thus greatly simplifying the experimental paradigm that arises when overexpressing proteins or downregulating gene expression. We have generated CRISPR tools to introduce double-strand breaks in the first coding exon of SNCA gene. Human iPSCs from a SNCA triplication carrier were growth adapted to single cell cloning and were transfected with the CRISPR constructs several rounds before genotyping of individual clones. We generated 11 clones with different mutant alleles relating to 4 knock-out (KO), 3KO, 2KO, 1 KO. The resulting iPSCs were karyotypically normal and expressed pluripotency markers. mRNA expression decreased corresponding to the number of functional copies of the SNCA gene. Dopaminergic neurons derived from these isogenic lines are analyzed for viability, differentiation potential and morphological as well as physiological changes to evaluate the effect of different 'gene doses' of alpha-synuclein. Here, we present a unique in vitro model system to study the impact of a-syn in an isogenic background. This system will be extremely useful for the study of a-syn associated pathways, drug screening, and the pharmacological modulation of a-syn levels in PD pathophysiology.