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Sci Rep. 2016 Dec 2;6:38198. doi: 10.1038/srep38198.

Improved bi-allelic modification of a transcriptionally silent locus in patient-derived iPSC by Cas9 nickase.

Author information

1
Research Group Translational Hepatology and Stem Cell Biology, Cluster of Excellence REBIRTH, Hannover Medical School, Hannover, 30625, Germany.
2
Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Hannover, 30362, Germany.
3
Institute of Experimental Hematology, Cluster of Excellence REBIRTH, Hannover Medical School, Hannover, 30625, Germany.
4
Department of Neurology, Hannover Medical School, Hannover, 30625, Germany.
5
Max Planck Institute for Infection Biology, Department of Regulation in Infection Biology, Berlin, 10117, Germany.
6
The Laboratory for Molecular Infection Medicine Sweden (MIMS), Umeå Centre for Microbial Research (UCMR), Department of Molecular Biology, Umeå University, Umeå, 90187, Sweden.
7
Center for Chronic Immunodeficiency, Medical Center - University of Freiburg, Freiburg, 79106, Germany.
8
Institute for Cell and Gene Therapy, Medical Center - University of Freiburg, Freiburg, 79106, Germany.
9
Faculty of Medicine, University of Freiburg, Freiburg, 79106, Germany.
10
Institute of Human Genetics, Hannover Medical School, Hannover, 30625, Germany.
11
Max Planck Institute for Molecular Biomedicine, Cell and Developmental Biology, Münster, 48149, Germany.

Abstract

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.

PMID:
27910942
PMCID:
PMC5133597
DOI:
10.1038/srep38198
[Indexed for MEDLINE]
Free PMC Article

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