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

Peter Barbuti, Paul Antony, Bruno Santos, François Massart, Gérald Cruciani, Claire Dording, Jonathan Arias, Jens Schwamborn, Rejko Krüger

Research output: Contribution to journalArticleResearchpeer-review

10 Citations (Scopus)

Abstract

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.

Original languageEnglish
JournalCells
Volume9
Issue number9
DOIs
Publication statusPublished - 10 Sep 2020

Keywords

  • A30P
  • CRISPR-Cas9
  • Parkinson’s disease (PD), patient-derived iPS
  • SNCA
  • alpha-synuclein
  • fluorescent-activated cell sorting (FACS)
  • high-content screening (HCS)
  • isogenic cell lines
  • single-cell clones

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