title: Efficient designer nuclease-based homologous recombination enables direct PCR screening for footprintless targeted human pluripotent stem cells.
abstract: Genetic engineering of human induced pluripotent stem cells (hiPSCs) via customized designer nucleases has been shown to be significantly more efficient than conventional gene targeting, but still typically depends on the introduction of additional genetic selection elements. In our study, we demonstrate the efficient nonviral and selection-independent gene targeting in human pluripotent stem cells (hPSCs). Our high efficiencies of up to 1.6% of gene-targeted hiPSCs, accompanied by a low background of randomly inserted transgenes, eliminated the need for antibiotic or fluorescence-activated cell sorting selection, and allowed the use of short donor oligonucleotides for footprintless gene editing. Gene-targeted hiPSC clones were established simply by direct PCR screening. This optimized approach allows targeted transgene integration into safe harbor sites for more predictable and robust expression and enables the straightforward generation of disease-corrected, patient-derived iPSC lines for research purposes and, ultimately, for future clinical applications.
authors.affiliation: Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Department of Cardiac, Thoracic, Transplant and Vascular Surgery, Hannover Medical School, 30625 Hannover, Germany ; REBIRTH-Cluster of Excellence, Hannover Medical School, 30625 Hannover, Germany.
authors.forename: Sylvia
authors.initials: S
authors.lastname: Merkert
authors.affiliation: Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Department of Cardiac, Thoracic, Transplant and Vascular Surgery, Hannover Medical School, 30625 Hannover, Germany ; REBIRTH-Cluster of Excellence, Hannover Medical School, 30625 Hannover, Germany.
authors.forename: Stephanie
authors.initials: S
authors.lastname: Wunderlich
authors.affiliation: Institute for Cell and Gene Therapy, University Medical Center Freiburg, 79106 Freiburg, Germany ; Center for Chronic Immunodeficiency, University Medical Center Freiburg, 79106 Freiburg, Germany ; Institute of Experimental Hematology, Hannover Medical School, 30625 Hannover, Germany.
authors.forename: Christien
authors.initials: C
authors.lastname: Bednarski
authors.affiliation: Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Department of Cardiac, Thoracic, Transplant and Vascular Surgery, Hannover Medical School, 30625 Hannover, Germany ; REBIRTH-Cluster of Excellence, Hannover Medical School, 30625 Hannover, Germany.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Beier
authors.affiliation: Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Department of Cardiac, Thoracic, Transplant and Vascular Surgery, Hannover Medical School, 30625 Hannover, Germany ; REBIRTH-Cluster of Excellence, Hannover Medical School, 30625 Hannover, Germany.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Haase
authors.affiliation: Institute of Experimental Hematology, Hannover Medical School, 30625 Hannover, Germany.
authors.forename: Anne-Kathrin
authors.initials: AK
authors.lastname: Dreyer
authors.affiliation: Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Department of Cardiac, Thoracic, Transplant and Vascular Surgery, Hannover Medical School, 30625 Hannover, Germany ; REBIRTH-Cluster of Excellence, Hannover Medical School, 30625 Hannover, Germany.
authors.forename: Kristin
authors.initials: K
authors.lastname: Schwanke
authors.affiliation: Institute of Experimental Hematology, Hannover Medical School, 30625 Hannover, Germany.
authors.forename: Johann
authors.initials: J
authors.lastname: Meyer
authors.affiliation: Institute of Cell and Molecular Pathology, Hannover Medical School, 30625 Hannover, Germany.
authors.forename: Gudrun
authors.initials: G
authors.lastname: Göhring
authors.affiliation: Institute for Cell and Gene Therapy, University Medical Center Freiburg, 79106 Freiburg, Germany ; Center for Chronic Immunodeficiency, University Medical Center Freiburg, 79106 Freiburg, Germany ; Institute of Experimental Hematology, Hannover Medical School, 30625 Hannover, Germany.
authors.forename: Toni
authors.initials: T
authors.lastname: Cathomen
authors.affiliation: Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Department of Cardiac, Thoracic, Transplant and Vascular Surgery, Hannover Medical School, 30625 Hannover, Germany ; REBIRTH-Cluster of Excellence, Hannover Medical School, 30625 Hannover, Germany.
authors.forename: Ulrich
authors.initials: U
authors.lastname: Martin
mesh_terms: D002478:Cells, Cultured; D059766:DNA End-Joining Repair; D004720:Endonucleases / Q000378:metabolism*; D055786:Gene Knockout Techniques; D018390:Gene Targeting; D056426:Genetic Loci; D059765:Homologous Recombination; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D039904:Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism*; D016133:Polymerase Chain Reaction
chemical_list: D009838:Oligodeoxyribonucleotides; D004720:Endonucleases