FACS-Assisted CRISPR-Cas9 Genome Editing Facilitates Parkinson's Disease Modeling

• Published in: Stem cell reports Vol. 9; no. 5; pp. 1423 - 1431
• Main Authors: Arias-Fuenzalida, Jonathan, Jarazo, Javier, Qing, Xiaobing, Walter, Jonas, Gomez-Giro, Gemma, Nickels, Sarah Louise, Zaehres, Holm, Schöler, Hans Robert, Schwamborn, Jens Christian
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 14-11-2017; Elsevier
• Subjects: Alleles; alpha-Synuclein - genetics; Cells, Cultured; CRISPR-Cas Systems; DNA End-Joining Repair - genetics; FACS; Flow Cytometry - methods; Gene Editing - methods; genome editing; Humans; Induced Pluripotent Stem Cells - cytology; Induced Pluripotent Stem Cells - metabolism; mitochondria; neuro stem cell; Parkinson Disease - genetics; random integration; repetitive elements; stem cells; α-synuclein; stem cells; genome editing; mitochondria; repetitive elements; FACS; random integration; neuro stem cell; α-synuclein
• ISSN: 2213-6711; 2213-6711
• DOI: 10.1016/j.stemcr.2017.08.026

Genome editing and human induced pluripotent stem cells hold great promise for the development of isogenic disease models and the correction of disease-associated mutations for isogenic tissue therapy. CRISPR-Cas9 has emerged as a versatile and simple tool for engineering human cells for such purposes. However, the current protocols to derive genome-edited lines require the screening of a great number of clones to obtain one free of random integration or on-locus non-homologous end joining (NHEJ)-containing alleles. Here, we describe an efficient method to derive biallelic genome-edited populations by the use of fluorescent markers. We call this technique FACS-assisted CRISPR-Cas9 editing (FACE). FACE allows the derivation of correctly edited polyclones carrying a positive selection fluorescent module and the exclusion of non-edited, random integrations and on-target allele NHEJ-containing cells. We derived a set of isogenic lines containing Parkinson's-disease-associated mutations in α-synuclein and present their comparative phenotypes.
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•Fluorescent protein-SNP pairs enable deterministic genotypes for genome editing•Repetitive elements modulate off-targeting random integration•Parkinson's disease NESCs present impaired mitochondrial energy performance
In this article, Arias-Fuenzalida and colleagues show a platform to achieve deterministic genotypes for genome editing. The combinatorial use of fluorescent proteins and defined SNPs facilitates the genome editing endeavor. Using neuroepithelial stem cells, they demonstrate early mitochondrial phenotypes in SNCA mutants.