期刊
STEM CELL REPORTS
卷 9, 期 5, 页码 1423-1431出版社
CELL PRESS
DOI: 10.1016/j.stemcr.2017.08.026
关键词
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资金
- LCSB Pluripotent Stem Cell Core Facility
- FNR (AFR, Aides a la Formation-Recherche)
- NCL-Stiftung
- Pelican award from the Fondation du Pelican de Mie et Pierre Hippert-Faber
- EU Joint Programme-Neurodegenerative Disease Research (JPND) project [INTER/JPND/14/02, INTER/JPND/15/11092422]
- European Union's Horizon Research and Innovation Program [668738]
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 a-synuclein and present their comparative phenotypes.
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