title: Genome-wide Specificity of Highly Efficient TALENs and CRISPR/Cas9 for T Cell Receptor Modification.
abstract: In T cells with transgenic high-avidity T cell receptors (TCRs), endogenous and transferred TCR chains compete for surface expression and may pair inappropriately, potentially causing autoimmunity. To knock out endogenous TCR expression, we assembled 12 transcription activator-like effector nucleases (TALENs) and five guide RNAs (gRNAs) from the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas9) system. Using TALEN mRNA, TCR knockout was successful in up to 81% of T cells. Additionally, we were able to verify targeted gene addition of a GFP gene by homology-directed repair at the TALEN target site, using a donor suitable for replacement of the reporter transgene with therapeutic TCR chains. Remarkably, analysis of TALEN and CRISPR/Cas9 specificity using integrase-defective lentiviral vector capture revealed only one off-target site for one of the gRNAs and three off-target sites for both of the TALENs, indicating a high level of specificity. Collectively, our work shows highly efficient and specific nucleases for T cell engineering.
authors.affiliation: Department of Translational Oncology, National Center for Tumor Diseases and German Cancer Research Center, 69120 Heidelberg, Germany.
authors.forename: Friederike
authors.initials: F
authors.lastname: Knipping
authors.affiliation: Division of Blood and Marrow Transplantation, Department of Pediatrics, University of Minnesota, Minneapolis, MN 55455, USA; Center for Genome Engineering, University of Minnesota, Minneapolis, MN 55455, USA; Stem Cell Institute, University of Minnesota, Minneapolis, MN 55455, USA; Asan-Minnesota Institute for Innovating Transplantation, Seoul 05505, Republic of Korea.
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Osborn
authors.affiliation: Department of Translational Oncology, National Center for Tumor Diseases and German Cancer Research Center, 69120 Heidelberg, Germany.
authors.forename: Karl
authors.initials: K
authors.lastname: Petri
authors.affiliation: Division of Blood and Marrow Transplantation, Department of Pediatrics, University of Minnesota, Minneapolis, MN 55455, USA; Center for Genome Engineering, University of Minnesota, Minneapolis, MN 55455, USA; Stem Cell Institute, University of Minnesota, Minneapolis, MN 55455, USA; Asan-Minnesota Institute for Innovating Transplantation, Seoul 05505, Republic of Korea.
authors.forename: Jakub
authors.initials: J
authors.lastname: Tolar
authors.affiliation: Department of Translational Oncology, National Center for Tumor Diseases and German Cancer Research Center, 69120 Heidelberg, Germany.
authors.forename: Hanno
authors.initials: H
authors.lastname: Glimm
authors.affiliation: Department of Translational Oncology, National Center for Tumor Diseases and German Cancer Research Center, 69120 Heidelberg, Germany.
authors.forename: Christof
authors.initials: C
authors.lastname: von Kalle
authors.affiliation: Department of Translational Oncology, National Center for Tumor Diseases and German Cancer Research Center, 69120 Heidelberg, Germany.
authors.forename: Manfred
authors.initials: M
authors.lastname: Schmidt
authors.affiliation: Department of Translational Oncology, National Center for Tumor Diseases and German Cancer Research Center, 69120 Heidelberg, Germany.
authors.forename: Richard
authors.initials: R
authors.lastname: Gabriel
keywords: T cell therapy; gene editing; off target