title: TALENs facilitate targeted genome editing in human cells with high specificity and low cytotoxicity.
abstract: Designer nucleases have been successfully employed to modify the genomes of various model organisms and human cell types. While the specificity of zinc-finger nucleases (ZFNs) and RNA-guided endonucleases has been assessed to some extent, little data are available for transcription activator-like effector-based nucleases (TALENs). Here, we have engineered TALEN pairs targeting three human loci (CCR5, AAVS1 and IL2RG) and performed a detailed analysis of their activity, toxicity and specificity. The TALENs showed comparable activity to benchmark ZFNs, with allelic gene disruption frequencies of 15-30% in human cells. Notably, TALEN expression was overall marked by a low cytotoxicity and the absence of cell cycle aberrations. Bioinformatics-based analysis of designer nuclease specificity confirmed partly substantial off-target activity of ZFNs targeting CCR5 and AAVS1 at six known and five novel sites, respectively. In contrast, only marginal off-target cleavage activity was detected at four out of 49 predicted off-target sites for CCR5- and AAVS1-specific TALENs. The rational design of a CCR5-specific TALEN pair decreased off-target activity at the closely related CCR2 locus considerably, consistent with fewer genomic rearrangements between the two loci. In conclusion, our results link nuclease-associated toxicity to off-target cleavage activity and corroborate TALENs as a highly specific platform for future clinical translation.
authors.affiliation: Institute for Cell and Gene Therapy, University Medical Center Freiburg, 79106 Freiburg, Germany Center for Chronic Immunodeficiency, University Medical Center Freiburg, 79108 Freiburg, Germany Institute of Experimental Hematology, Hannover Medical School, 30625 Hannover, Germany.
authors.forename: Claudio
authors.initials: C
authors.lastname: Mussolino
authors.affiliation: Institute for Cell and Gene Therapy, University Medical Center Freiburg, 79106 Freiburg, Germany Center for Chronic Immunodeficiency, University Medical Center Freiburg, 79108 Freiburg, Germany Institute of Experimental Hematology, Hannover Medical School, 30625 Hannover, Germany.
authors.forename: Jamal
authors.initials: J
authors.lastname: Alzubi
authors.affiliation: Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA 30332, USA.
authors.forename: Eli J
authors.initials: EJ
authors.lastname: Fine
authors.affiliation: Institute of Genetics, Ludwig-Maximilians-University Munich, 82152 Martinsried, Germany.
authors.forename: Robert
authors.initials: R
authors.lastname: Morbitzer
authors.affiliation: Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA 30332, USA.
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Cradick
authors.affiliation: Institute of Genetics, Ludwig-Maximilians-University Munich, 82152 Martinsried, Germany Center for Plant Molecular Biology, Eberhard-Karls-University, 72076 Tübingen, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Lahaye
authors.affiliation: Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA 30332, USA.
authors.forename: Gang
authors.initials: G
authors.lastname: Bao
authors.affiliation: Institute for Cell and Gene Therapy, University Medical Center Freiburg, 79106 Freiburg, Germany Center for Chronic Immunodeficiency, University Medical Center Freiburg, 79108 Freiburg, Germany Institute of Experimental Hematology, Hannover Medical School, 30625 Hannover, Germany toni.cathomen@uniklinik-freiburg.de.
authors.forename: Toni
authors.initials: T
authors.lastname: Cathomen
mesh_terms: D002478:Cells, Cultured; D053837:DNA Cleavage; D003851:Deoxyribonucleases / Q000737:chemistry / Q000378:metabolism*; D056426:Genetic Loci; D015894:Genome, Human; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D053631:Interleukin Receptor Common gamma Subunit / Q000235:genetics; D015202:Protein Engineering; D019713:Receptors, CCR5 / Q000235:genetics
chemical_list: C584933:CCR5 protein, human; C507941:IL2RG protein, human; D053631:Interleukin Receptor Common gamma Subunit; D019713:Receptors, CCR5; D003851:Deoxyribonucleases