Luxembourg Centre for Systems Biomedicine (LCSB), Developmental and Cellular Biology, University of Luxembourg, 7 Avenue des Hauts-Fourneaux, Luxembourg City 4362, Luxembourg; Graduate School of Biostudies, Kyoto University, Kyoto 606-8502, Japan.
Luxembourg Centre for Systems Biomedicine (LCSB), Developmental and Cellular Biology, University of Luxembourg, 7 Avenue des Hauts-Fourneaux, Luxembourg City 4362, Luxembourg.
Stem Cell Reports. 2017 Nov 14;9(5):1423-1431. doi: 10.1016/j.stemcr.2017.08.026. Epub 2017 Oct 5.
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.
基因组编辑和人类诱导多能干细胞为同源疾病模型的发展和同源组织治疗中疾病相关突变的校正带来了巨大的希望。CRISPR-Cas9 已经成为一种用于工程人类细胞的多功能和简单工具。然而，目前衍生基因组编辑系的方案需要筛选大量的克隆以获得不含随机整合或靶内非同源末端连接（NHEJ）的等位基因。在这里，我们描述了一种通过荧光标记物衍生双等位基因基因组编辑群体的有效方法。我们将该技术称为 FACS 辅助 CRISPR-Cas9 编辑（FACE）。FACE 允许衍生携带阳性选择荧光模块的正确编辑的多克隆，并排除非编辑的、随机整合的和靶标等位基因 NHEJ 包含的细胞。我们衍生了一组含有帕金森病相关突变的α-突触核蛋白的同源系，并呈现了它们的比较表型。
