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

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.

Gene-corrected Parkinson’s disease neurons show the A30P alpha-synuclein point mutation leads to reduced neuronal branching and function

“…The generation and characterisation of induced pluripotent stem cells (iPSCs) from the dermal fibroblasts has been described 38 and has a unique identifier HIHDNDi001-B (https://hpscreg.eu/cell-line/HIHDNDi001-B). The generation of single-cell gene-corrected patient-derived iPS clones from the A30P patient has also been described 35 . Ethical approval was obtained from the National Committee for Ethics in Research, Luxembourg (Comité National d’Ethique dans la Recherche; CNER #201411/05).…”

“…The generation of ventral midbrain dopaminergic (vmDA) neurons was performed according to the protocol elsewhere described 39 . The vmDA neurons were generated via a multipotent neuronal precursor cell (NPC) stage, the generation and characterisation of the NPCs and vmDA neurons used this study has been previously described 35 .…”

“…The neurons were plated at 200,000 cells/coverslip with the addition of 10 μM Rho-Kinase Inhibitor Y-27632 (10 μM; Abcam, Cambridge, UK; Ab120129). After 48 hours, the neurons were fixed and stained as previously described 38 using primary antibodies for β-3-Tubulin (TUJ1; 1:200; BioLegend, San Diego, CA, USA; 801201) and TH (1:300; Millipore, Burlington, MA, USA; AB152) 35 . For the image acquisition, 10 to 12 Z-stack images per coverslip were acquired using Zeiss spinning disk confocal microscope from four independent directed neuronal differentiations.…”

“…Not all cells will be edited and fluorescent reporters, antibiotic resistance or a combination of both, are used to enrich and/or sort out the edited cells to generate the isogenic cell line 32–34 . Previously, we showed that with the aid of high-content screening (HCS) technology we were able to generate multiple single-cell gene-corrected patient-derived iPS cell clones from a PD patient harbouring the pathogenic p.A30P mutation in SNCA 35 . The advantage of having single-cell clones is that it provides the guarantee that every cell in that colony and expanded cell line is edited and gene-corrected.…”

CRISPR Genome Editing: How to Make a Fantastic Method Even Better

“…If CRISPR genome editing is used on patient-derived tissue specific stem cells or induced pluripotent stem (iPS) cells, only successfully repaired cells should be transplanted back to the patient. To facilitate the selection of such cells, Barbuti et al present a high-content screening approach to identify repaired iPS cells obtained from a Parkinson patient [ 8 ]. The ability of the gRNA–Cas9 riboprotein to bind to a unique site in the genome can be used for the targeted introduction of DSB, and to transport chromatin-modifying enzymes to that genomic site.…”