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Eggenschwiler, Reto; Moslem, Mohsen; Fráguas, Mariane Serra; Galla, Melanie; Papp, Oliver; Naujock, Maximilian; Fonfara, Ines; Gensch, Ingrid; Wähner, Annabell; Beh-Pajooh, Abbas; Mussolino, Claudio; Tauscher, Marcel; Steinemann, Doris; Wegner, Florian; Petri, Susanne; Schambach, Axel; Charpentier, Emmanuelle; Cathomen, Toni; Cantz, Tobias
Scientific reports, 12/2016, Letnik: 6, Številka: 1Journal Article
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 puroΔtk 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.
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JCR | SNIP | JCR | SNIP | JCR | SNIP | JCR | SNIP |
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in: SICRIS
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