Genome-wide Specificity of Highly Efficient TALENs and CRISPR/Cas9 for T Cell Receptor Modification

Friederike Knipping; Mark J. Osborn; Karl Petri; Jakub Tolar; Hanno Glimm; Christof von Kalle; Manfred Schmidt; Richard Gabriel

doi:10.1016/j.omtm.2017.01.005

Genome-wide Specificity of Highly Efficient TALENs and CRISPR/Cas9 for T Cell Receptor Modification

Friederike Knipping, Mark J. Osborn, Karl Petri, Jakub Tolar, Hanno Glimm, Christof von Kalle, Manfred Schmidt, Richard Gabriel

Research output: Contribution to journal › Article › peer-review

30 Scopus citations

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.

Original language	English (US)
Pages (from-to)	213-224
Number of pages	12
Journal	Molecular Therapy - Methods and Clinical Development
Volume	4
DOIs	https://doi.org/10.1016/j.omtm.2017.01.005
State	Published - Mar 17 2017

Bibliographical note

Funding Information:
We thank Raffaele Fronza for helpful discussions. This work was supported by the European Union Seventh Framework Program (grant 601958-SUPERSIST) and NCT3.0 funding program (NCT3.0_2015.13 ImmunOmics). M.J.O. and J.T. are also thankful for the generosity of the Lindahl family, the Children's Cancer Research Fund, and the Corrigan family. M.J.O. is supported by 8UL1TR000114-02. J.T. is supported in part by R01 AR063070 and P01 CA065493. The present work was partly supported by research funds from the National Research Foundation of Korea (grant NRF-2015K1A4A3046807). Research reported in this publication was supported by the National Center for Advancing Translational Sciences of the National Institutes of Health (award UL1TR000114 to M.J.O.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Publisher Copyright:
© 2017 The Authors

Keywords

T cell therapy
gene editing
off target

Publisher link

10.1016/j.omtm.2017.01.005

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5363317

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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.",

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note = "Funding Information: We thank Raffaele Fronza for helpful discussions. This work was supported by the European Union Seventh Framework Program (grant 601958-SUPERSIST) and NCT3.0 funding program (NCT3.0_2015.13 ImmunOmics). M.J.O. and J.T. are also thankful for the generosity of the Lindahl family, the Children's Cancer Research Fund, and the Corrigan family. M.J.O. is supported by 8UL1TR000114-02. J.T. is supported in part by R01 AR063070 and P01 CA065493. The present work was partly supported by research funds from the National Research Foundation of Korea (grant NRF-2015K1A4A3046807). Research reported in this publication was supported by the National Center for Advancing Translational Sciences of the National Institutes of Health (award UL1TR000114 to M.J.O.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Publisher Copyright: {\textcopyright} 2017 The Authors",

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AU - Osborn, Mark J.

AU - Petri, Karl

AU - Tolar, Jakub

AU - Glimm, Hanno

AU - von Kalle, Christof

AU - Schmidt, Manfred

AU - Gabriel, Richard

N1 - Funding Information: We thank Raffaele Fronza for helpful discussions. This work was supported by the European Union Seventh Framework Program (grant 601958-SUPERSIST) and NCT3.0 funding program (NCT3.0_2015.13 ImmunOmics). M.J.O. and J.T. are also thankful for the generosity of the Lindahl family, the Children's Cancer Research Fund, and the Corrigan family. M.J.O. is supported by 8UL1TR000114-02. J.T. is supported in part by R01 AR063070 and P01 CA065493. The present work was partly supported by research funds from the National Research Foundation of Korea (grant NRF-2015K1A4A3046807). Research reported in this publication was supported by the National Center for Advancing Translational Sciences of the National Institutes of Health (award UL1TR000114 to M.J.O.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Publisher Copyright: © 2017 The Authors

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N2 - 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.

AB - 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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ER -

Genome-wide Specificity of Highly Efficient TALENs and CRISPR/Cas9 for T Cell Receptor Modification

Abstract

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