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: | , , , , , |
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| Dokumenttyp: | Article (Journal) |
| Sprache: | Englisch |
| Veröffentlicht: |
12 February 2017
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| 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/ 
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| Verfasserangaben: | Friederike Knipping, Mark J. Osborn, Karl Petri, Jakub Tolar, Hanno Glimm, Christof von Kalle, Manfred Schmidt, and Richard Gabriel |
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| 245 | 1 | 0 | |a Genome-wide specificity of highly efficient TALENs and CRISPR/Cas9 for T cell receptor modification |c Friederike Knipping, Mark J. Osborn, Karl Petri, Jakub Tolar, Hanno Glimm, Christof von Kalle, Manfred Schmidt, and Richard Gabriel |
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| 520 | |a 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. | ||
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