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Improved bi-allelic modification of a transcriptionally silent locus in patient-derived iPSC by Cas9 nickase
- Eggenschwiler, Reto (author)
- Moslem, Mohsen (author)
- Fráguas, Mariane Serra (author)
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- Galla, Melanie (author)
- Papp, Oliver (author)
- Naujock, Maximilian (author)
- Gensch, Ingrid (author)
- Wähner, Annabell (author)
- Beh-Pajooh, Abbas (author)
- Mussolino, Claudio (author)
- Tauscher, Marcel (author)
- Steinemann, Doris (author)
- Wegner, Florian (author)
- Petri, Susanne (author)
- Schambach, Axel (author)
- Cathomen, Toni (author)
- Cantz, Tobias (author)
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- (creator_code:org_t)
- 2016-12-02
- 2016
- English.
- In: Scientific Reports. - : Nature Publishing Group. - 2045-2322. ; 6
- Journal article (peer-reviewed)
Abstract
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- Homology directed repair (HDR)-based genome editing via selectable long flanking arm donors can be hampered by local transgene silencing at transcriptionally silent loci. Here, we report efficient bi-allelic modification of a silent locus in patient-derived hiPSC by using Cas9 nickase and a silencing-resistant donor construct that contains an excisable selection/counter-selection cassette. To identify the most active single guide RNA (sgRNA)/nickase combinations, we employed a lentiviral vector-based reporter assay to determine the HDR efficiencies in cella. Next, we used the most efficient pair of sgRNAs for targeted integration of an improved, silencing-resistant plasmid donor harboring a piggyBac-flanked puro Delta tk cassette. Moreover, we took advantage of a dual-fluorescence selection strategy for bi-allelic targeting and achieved 100% counter-selection efficiency after bi-allelic excision of the selection/counter-selection cassette. Together, we present an improved system for efficient bi-allelic modification of transcriptionally silent loci in human pluripotent stem cells.
Subject headings
- MEDICIN OCH HÄLSOVETENSKAP -- Medicinska och farmaceutiska grundvetenskaper -- Mikrobiologi inom det medicinska området (hsv//swe)
- MEDICAL AND HEALTH SCIENCES -- Basic Medicine -- Microbiology in the medical area (hsv//eng)
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- Fonfara, Ines
- Gensch, Ingrid
- Wähner, Annabell
- Beh-Pajooh, Abba ...
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- Tauscher, Marcel
- Steinemann, Dori ...
- Wegner, Florian
- Petri, Susanne
- Schambach, Axel
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- Cathomen, Toni
- Cantz, Tobias
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