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          Institute: MPI für molekulare Biomedizin     Collection: Jahrbuch 2018 (publ. 2017, arch)     Display Documents



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ID: 744168.0, MPI für molekulare Biomedizin / Jahrbuch 2018 (publ. 2017, arch)
FACS-Assisted CRISPR-Cas9 Genome Editing Facilitates Parkinson's Disease Modeling
Authors:Arias-Fuenzalida, J.; Jarazo, J.; Qing, X.; Walter, J.; Gomez-Giro, G.; Nickels, S. L.; Zaehres, H.; Scholer, H. R.; Schwamborn, J. C.
Date of Publication (YYYY-MM-DD):2017-11-14
Title of Journal:Stem Cell Reports
Volume:9
Issue / Number:5
Start Page:1423
End Page:1431
Review Status:Internal review
Audience:Not Specified
Abstract / Description: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 alpha-synuclein and present their comparative phenotypes.
Free Keywords:Alleles; *CRISPR-Cas Systems; Cells, Cultured; DNA End-Joining Repair/genetics; Flow Cytometry/methods; Gene Editing/*methods; Humans; Induced Pluripotent Stem Cells/cytology/*metabolism; Parkinson Disease/*genetics; alpha-Synuclein/*genetics; *facs; *genome editing; *mitochondria; *neuro stem cell; *random integration; *repetitive elements; *stem cells; *alpha-synuclein
External Publication Status:published
Document Type:Article
Communicated by:MPI für molekulare Biomedizin
Affiliations:MPI für molekulare Biomedizin
External Affiliations:Luxembourg Centre for Systems Biomedicine (LCSB), Developmental and Cellular Biology, University of Luxembourg, 7 Avenue des Hauts-Fourneaux, Luxembourg City 4362, Luxembourg. Luxembourg Centre for Systems Biomedicine (LCSB), Developmental and Cellular Biology, University of Luxembourg, 7 Avenue des Hauts-Fourneaux, Luxembourg City 4362, Luxembourg; Max Planck Institute for Molecular Biomedicine, Laboratory of Cell and Developmental Biology, Roentgenstrasse 20, Muenster, Germany. Luxembourg Centre for Systems Biomedicine (LCSB), Developmental and Cellular Biology, University of Luxembourg, 7 Avenue des Hauts-Fourneaux, Luxembourg City 4362, Luxembourg; Life Science Research Unit (LSRU), Systems Biology, University of Luxembourg, 6 Avenue du Swing, Luxembourg City 4367, Luxembourg. Max Planck Institute for Molecular Biomedicine, Laboratory of Cell and Developmental Biology, Roentgenstrasse 20, Muenster, Germany; Ruhr-University Bochum, Medical Faculty, Department of Anatomy and Molecular Embryology, 44801 Bochum, Germany. Max Planck Institute for Molecular Biomedicine, Laboratory of Cell and Developmental Biology, Roentgenstrasse 20, Muenster, Germany; Medical Faculty, Westphalian Wilhelms University Muenster, 48149 Muenster, Germany. Luxembourg Centre for Systems Biomedicine (LCSB), Developmental and Cellular Biology, University of Luxembourg, 7 Avenue des Hauts-Fourneaux, Luxembourg City 4362, Luxembourg. Electronic address: jens.schwamborn@uni.lu.
Identifiers:ISSN:2213-6711 (Electronic) 2213-6711 (Linking) %R 10.1... [ID No:1]
URL:https://www.ncbi.nlm.nih.gov/pubmed/28988985 [ID No:2]
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