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Genome-wide specificity of highly efficient TALENs and CRISPR/Cas9 for T cell receptor modification

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 nuclease...

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Hauptverfasser: Knipping, Friederike (VerfasserIn) , Petri, Karl (VerfasserIn) , Glimm, Hanno (VerfasserIn) , Kalle, Christof von (VerfasserIn) , Schmidt, Manfred (VerfasserIn) , Gabriel, Richard (VerfasserIn)
Dokumenttyp: Article (Journal)
Sprache:Englisch
Veröffentlicht: 12 February 2017
In: Molecular therapy. Methods & clinical development
Year: 2017, Jahrgang: 4, Pages: 213-224
ISSN:2329-0501
DOI:10.1016/j.omtm.2017.01.005
Online-Zugang:Verlag, kostenfrei, Volltext: http://dx.doi.org/10.1016/j.omtm.2017.01.005
Verlag, kostenfrei, Volltext: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5363317/
Volltext
Verfasserangaben:Friederike Knipping, Mark J. Osborn, Karl Petri, Jakub Tolar, Hanno Glimm, Christof von Kalle, Manfred Schmidt, and Richard Gabriel
Beschreibung
Zusammenfassung: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.
Beschreibung:Available online 12 February 2017
Gesehen am 04.09.2018
Beschreibung:Online Resource
ISSN:2329-0501
DOI:10.1016/j.omtm.2017.01.005

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