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Gene editing and clonal isolation of human induced pluripotent stem cells using CRISPR/Cas9

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Item Type:Article
Title:Gene editing and clonal isolation of human induced pluripotent stem cells using CRISPR/Cas9
Creators Name:Yumlu, S. and Stumm, J. and Bashir, S. and Dreyer, A.K. and Lisowski, P. and Danner, E. and Kühn, R.
Abstract:Human induced pluripotent stem cells (hiPSCs) represent an ideal in vitro platform to study human genetics and biology. The recent advent of programmable nucleases makes also the human genome amenable to experimental genetics through either the correction of mutations in patient-derived iPSC lines or the de novo introduction of mutations into otherwise healthy iPSCs. The production of specific and sometimes complex genotypes in multiple cell lines requires efficient and streamlined gene editing technologies. In this article we provide protocols for gene editing in hiPSCs. We presently achieve high rates of gene editing at up to three loci using a modified iCRISPR system. This system includes a doxycycline inducible Cas9 and sgRNA/reporter plasmids for the enrichment of transfected cells by fluorescence-activated cell sorting (FACS). Here we cover the selection of target sites, vector construction, transfection, and isolation and genotyping of modified hiPSC clones.
Keywords:Pluripotent Stem Cells, Gene Editing, CRISPR, Cas9, Knockout, Knockin
Source:Methods
ISSN:1046-2023
Publisher:Elsevier / Academic Press (U.S.A.)
Volume:121-122
Page Range:29-44
Date:15 May 2017
Additional Information:Copyright © 2017 Elsevier Inc. All rights reserved.
Official Publication:https://doi.org/10.1016/j.ymeth.2017.05.009
PubMed:View item in PubMed

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