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Article

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

1
Translational Neuroscience, Luxembourg Centre for Systems Biomedicine, University of Luxembourg, L-4362 Luxembourg, Luxembourg
2
Transversal Translational Medicine, Luxembourg Institute of Health, L-1445 Luxembourg, Luxembourg
3
Department of Pathology and Cell Biology, Columbia University Irving Medical Center, New York, NY 10032, USA
4
Developmental and Cellular Biology, Luxembourg Centre for Systems Biomedicine, University of Luxembourg, L-4362 Luxembourg, Luxembourg
5
Department of Biosciences and Nutrition, Neo, Karolinska Institutet, SE-141 83 Huddinge, Sweden
6
Department of Neurodegenerative diseases, Hertie Institute for Clinical Brain Research, University Clinics Tübingen, 72076 Tübingen, Germany
7
Parkinson Research Clinic, Centre Hospitalier de Luxembourg (CHL), L-1210 Luxembourg, Luxembourg
*
Authors to whom correspondence should be addressed.
Senior author.
Cells 2020, 9(9), 2065; https://doi.org/10.3390/cells9092065
Received: 14 August 2020 / Accepted: 8 September 2020 / Published: 10 September 2020
(This article belongs to the Special Issue CRISPR Genome Editing)
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 SNCA 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 SNCA 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. View Full-Text
Keywords: CRISPR-Cas9; high-content screening (HCS); fluorescent-activated cell sorting (FACS); Parkinson’s disease (PD), patient-derived iPS; single-cell clones; isogenic cell lines; SNCA; alpha-synuclein; A30P CRISPR-Cas9; high-content screening (HCS); fluorescent-activated cell sorting (FACS); Parkinson’s disease (PD), patient-derived iPS; single-cell clones; isogenic cell lines; SNCA; alpha-synuclein; A30P
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  • Externally hosted supplementary file 1
    Doi: 10.17881/lcsb.kcqg-tr55
    Description: A frozen page containing the source code required for the implementation of the CloneClassifier script with the output shown in Figure 3G is available.
MDPI and ACS Style

Barbuti, P.; Antony, P.; Santos, B.; Massart, F.; Cruciani, G.; Dording, C.; Arias, J.; Schwamborn, J.; Krüger, R. 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. Cells 2020, 9, 2065. https://doi.org/10.3390/cells9092065

AMA Style

Barbuti P, Antony P, Santos B, Massart F, Cruciani G, Dording C, Arias J, Schwamborn J, Krüger R. 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. Cells. 2020; 9(9):2065. https://doi.org/10.3390/cells9092065

Chicago/Turabian Style

Barbuti, Peter, Paul Antony, Bruno Santos, François Massart, Gérald Cruciani, Claire Dording, Jonathan Arias, Jens Schwamborn, and Rejko Krüger. 2020. "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" Cells 9, no. 9: 2065. https://doi.org/10.3390/cells9092065

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