title: Improved bi-allelic modification of a transcriptionally silent locus in patient-derived iPSC by Cas9 nickase.
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.
authors.affiliation: Research Group Translational Hepatology and Stem Cell Biology, Cluster of Excellence REBIRTH, Hannover Medical School, Hannover, 30625, Germany.|Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Hannover, 30362, Germany.
authors.forename: Reto
authors.initials: R
authors.lastname: Eggenschwiler
authors.affiliation: Research Group Translational Hepatology and Stem Cell Biology, Cluster of Excellence REBIRTH, Hannover Medical School, Hannover, 30625, Germany.|Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Hannover, 30362, Germany.
authors.forename: Mohsen
authors.initials: M
authors.lastname: Moslem
authors.affiliation: Research Group Translational Hepatology and Stem Cell Biology, Cluster of Excellence REBIRTH, Hannover Medical School, Hannover, 30625, Germany.|Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Hannover, 30362, Germany.
authors.forename: Mariane Serra
authors.initials: MS
authors.lastname: Fráguas
authors.affiliation: Institute of Experimental Hematology, Cluster of Excellence REBIRTH, Hannover Medical School, Hannover, 30625, Germany.
authors.forename: Melanie
authors.initials: M
authors.lastname: Galla
authors.affiliation: Research Group Translational Hepatology and Stem Cell Biology, Cluster of Excellence REBIRTH, Hannover Medical School, Hannover, 30625, Germany.|Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Hannover, 30362, Germany.
authors.forename: Oliver
authors.initials: O
authors.lastname: Papp
authors.affiliation: Department of Neurology, Hannover Medical School, Hannover, 30625, Germany.
authors.forename: Maximilian
authors.initials: M
authors.lastname: Naujock
authors.affiliation: Max Planck Institute for Infection Biology, Department of Regulation in Infection Biology, Berlin, 10117, Germany.|The Laboratory for Molecular Infection Medicine Sweden (MIMS), Umeå Centre for Microbial Research (UCMR), Department of Molecular Biology, Umeå University, Umeå, 90187, Sweden.
authors.forename: Ines
authors.initials: I
authors.lastname: Fonfara
authors.affiliation: Research Group Translational Hepatology and Stem Cell Biology, Cluster of Excellence REBIRTH, Hannover Medical School, Hannover, 30625, Germany.|Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Hannover, 30362, Germany.
authors.forename: Ingrid
authors.initials: I
authors.lastname: Gensch
authors.affiliation: Research Group Translational Hepatology and Stem Cell Biology, Cluster of Excellence REBIRTH, Hannover Medical School, Hannover, 30625, Germany.|Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Hannover, 30362, Germany.
authors.forename: Annabell
authors.initials: A
authors.lastname: Wähner
authors.affiliation: Research Group Translational Hepatology and Stem Cell Biology, Cluster of Excellence REBIRTH, Hannover Medical School, Hannover, 30625, Germany.|Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Hannover, 30362, Germany.
authors.forename: Abbas
authors.initials: A
authors.lastname: Beh-Pajooh
authors.affiliation: Center for Chronic Immunodeficiency, Medical Center - University of Freiburg, Freiburg, 79106, Germany.|Institute for Cell and Gene Therapy, Medical Center - University of Freiburg, Freiburg, 79106, Germany.|Faculty of Medicine, University of Freiburg, Freiburg, 79106, Germany.
authors.forename: Claudio
authors.initials: C
authors.lastname: Mussolino
authors.affiliation: Institute of Human Genetics, Hannover Medical School, Hannover, 30625, Germany.
authors.forename: Marcel
authors.initials: M
authors.lastname: Tauscher
authors.affiliation: Max Planck Institute for Molecular Biomedicine, Cell and Developmental Biology, Münster, 48149, Germany.
authors.forename: Doris
authors.initials: D
authors.lastname: Steinemann
authors.affiliation: Department of Neurology, Hannover Medical School, Hannover, 30625, Germany.
authors.forename: Florian
authors.initials: F
authors.lastname: Wegner
authors.affiliation: Department of Neurology, Hannover Medical School, Hannover, 30625, Germany.
authors.forename: Susanne
authors.initials: S
authors.lastname: Petri
authors.affiliation: Institute of Experimental Hematology, Cluster of Excellence REBIRTH, Hannover Medical School, Hannover, 30625, Germany.
authors.forename: Axel
authors.initials: A
authors.lastname: Schambach
authors.affiliation: Max Planck Institute for Infection Biology, Department of Regulation in Infection Biology, Berlin, 10117, Germany.|The Laboratory for Molecular Infection Medicine Sweden (MIMS), Umeå Centre for Microbial Research (UCMR), Department of Molecular Biology, Umeå University, Umeå, 90187, Sweden.
authors.forename: Emmanuelle
authors.initials: E
authors.lastname: Charpentier
authors.affiliation: Center for Chronic Immunodeficiency, Medical Center - University of Freiburg, Freiburg, 79106, Germany.|Institute for Cell and Gene Therapy, Medical Center - University of Freiburg, Freiburg, 79106, Germany.|Faculty of Medicine, University of Freiburg, Freiburg, 79106, Germany.
authors.forename: Toni
authors.initials: T
authors.lastname: Cathomen
authors.affiliation: Research Group Translational Hepatology and Stem Cell Biology, Cluster of Excellence REBIRTH, Hannover Medical School, Hannover, 30625, Germany.|Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Hannover, 30362, Germany.|Max Planck Institute for Molecular Biomedicine, Cell and Developmental Biology, Münster, 48149, Germany.
authors.forename: Tobias
authors.initials: T
authors.lastname: Cantz
mesh_terms: D000483:Alleles; D064113:CRISPR-Cas Systems; D020868:Gene Silencing; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000378:metabolism*; D014158:Transcription, Genetic