Using High-Content Screening to Generate Single-Cell Gene-Corrected Patient-Derived iPS Clones Reveals Excess Alpha-Synuclein with Familial Parkinson's Disease Point Mutation A30P

• Published in: Cells (Basel, Switzerland) Vol. 9; no. 9; p. 2065
• Main Authors: Barbuti, Peter, Antony, Paul, Santos, Bruno, Massart, François, Cruciani, Gérald, Dording, Claire, Arias, Jonathan, Schwamborn, Jens, Krüger, Rejko
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 10.09.2020; MDPI
• Subjects: alpha-Synuclein - genetics; Antibiotics; Cell Differentiation; Cell Line; Clonal selection; Clone Cells - cytology; Cloning; CRISPR; CRISPR-Cas9; Dopamine receptors; Drug resistance; Efficiency; Flow cytometry; fluorescent-activated cell sorting (FACS); Gene mutation; Genetic aspects; Genomes; Health aspects; high-content screening (HCS); Humans; Induced Pluripotent Stem Cells - cytology; Inhibitory postsynaptic potentials; isogenic cell lines; Karyotypes; Kinases; Medicin och hälsovetenskap; Mesencephalon; Movement disorders; Mutation; Neurodegenerative diseases; Parkinson disease; Parkinson Disease - genetics; Parkinson Disease - pathology; Parkinson's disease; Parkinson’s disease (PD), patient-derived iPS; Patients; Plasmids; Pluripotency; Point mutation; Proteins; single-cell clones; Stem cells; Synuclein; Luxembourg; St Louis Missouri; United States > US; Switzerland; Parkinson’s disease (PD), patient-derived iPS; single-cell clones; high-content screening (HCS); CRISPR-Cas9; isogenic cell lines; fluorescent-activated cell sorting (FACS); A30P; alpha-synuclein; SNCA
• ISSN: 2073-4409; 2073-4409
• DOI: 10.3390/cells9092065

The generation of isogenic induced pluripotent stem cell (iPSC) lines using CRISPR-Cas9 technology is a technically challenging, time-consuming process with variable efficiency. Here we use fluorescence-activated cell sorting (FACS) to sort biallelic CRISPR-Cas9 edited single-cell iPSC clones into high-throughput 96-well microtiter plates. We used high-content screening (HCS) technology and generated an in-house developed algorithm to select the correctly edited isogenic clones for continued expansion and validation. In our model we have gene-corrected the iPSCs of a Parkinson's disease (PD) patient carrying the autosomal dominantly inherited heterozygous c.88G>C mutation in the gene, which leads to the pathogenic p.A30P form of the alpha-synuclein protein. Undertaking a PCR restriction-digest mediated clonal selection strategy prior to sequencing, we were able to post-sort validate each isogenic clone using a quadruple screening strategy prior to generating footprint-free isogenic iPSC lines, retaining a normal molecular karyotype, pluripotency and three germ-layer differentiation potential. Directed differentiation into midbrain dopaminergic neurons revealed that expression is reduced in the gene-corrected clones, which was validated by a reduction at the alpha-synuclein protein level. The generation of single-cell isogenic clones facilitates new insights in the role of alpha-synuclein in PD and furthermore is applicable across patient-derived disease models.