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Gene editing and clonal isolation of human induced pluripotent stem cells using CRISPR/Cas9.
Methods 121-122, 29-44 (2017)
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.
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Publication type
Article: Journal article
Document type
Scientific Article
Keywords
Crispr ; Cas9 ; Gene Editing ; Knockin ; Knockout ; Pluripotent Stem Cells; Human Ips Cells; Crispr-cas9 Nucleases; Genome; Mutations; Specificity; Expression; Efficiency; Talens; Cas9; Dna
ISSN (print) / ISBN
1046-2023
e-ISSN
1095-9130
Journal
Methods
Quellenangaben
Volume: 121-122,
Pages: 29-44
Publisher
Elsevier
Publishing Place
Amsterdam [u.a.]
Non-patent literature
Publications
Reviewing status
Peer reviewed
Institute(s)
Institute of Developmental Genetics (IDG)