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p53 Hinders CRISPR/Cas9-Mediated Targeted Gene Disruption in Memory CD8 T Cells In Vivo

Samarchith P. Kurup, Steven J. Moioffer, Lecia L. Pewe and John T. Harty
J Immunol October 15, 2020, 205 (8) 2222-2230; DOI: https://doi.org/10.4049/jimmunol.2000654
Samarchith P. Kurup
*Department of Cellular Biology, University of Georgia, Athens, GA 30602;
†Center for Tropical and Emerging Global Diseases, University of Georgia, Athens, GA 30602;
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Steven J. Moioffer
‡Department of Pathology, University of Iowa, Iowa City, IA 52242; and
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Lecia L. Pewe
‡Department of Pathology, University of Iowa, Iowa City, IA 52242; and
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John T. Harty
‡Department of Pathology, University of Iowa, Iowa City, IA 52242; and
§Interdisciplinary Graduate Program in Immunology, University of Iowa, Iowa City, IA 52242
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Key Points

  • p53 precludes Cas9-mediated gene disruption in memory CD8 T cells.

  • Temporarily subduing p53 enables gene editing in memory CD8 T cells in vivo.

  • Cas9-mediated gene ablations in memory CD8 T cells are retained through recall.

Abstract

CRISPR/Cas9 technology has revolutionized rapid and reliable gene editing in cells. Although many cell types have been subjected to CRISPR/Cas9-mediated gene editing, there is no evidence of success in genetic alteration of Ag-experienced memory CD8 T cells. In this study, we show that CRISPR/Cas9-mediated gene editing in memory CD8 T cells precludes their proliferation after Ag re-encounter in vivo. This defect is mediated by the proapoptotic transcription factor p53, a sensor of DNA damage. Temporarily inhibiting p53 function offers a window of opportunity for the memory CD8 T cells to repair the DNA damage, facilitating robust recall responses on Ag re-encounter. We demonstrate this by functionally altering memory CD8 T cells using CRISPR/Cas9-mediated targeted gene disruption under the aegis of p53siRNA in the mouse model. Our approach thus adapts the CRISPR/Cas9 technology for memory CD8 T cells to undertake gene editing in vivo, for the first time, to our knowledge.

This article is featured in Top Reads, p.1979

Footnotes

  • This work was supported by National Institute of Allergy and Infectious Diseases, National Institutes of Health Grants AI42767, AI085515, AI114543, and AI100527 (to J.T.H.) and a University of Georgia Research Foundation startup grant (to S.P.K.).

  • The online version of this article contains supplemental material.

  • Abbreviations used in this article:

    IfngKO
    IFN-γ–deficient
    LCMV
    lymphocytic choriomeningitis virus
    Lm-Ova
    Listeria monocytogenes expressing chicken OVA
    rCas9
    recombinant Cas9
    RNP
    ribonucleoprotein
    sgRNA
    single-guide RNA.

  • Received June 4, 2020.
  • Accepted August 11, 2020.
  • Copyright © 2020 by The American Association of Immunologists, Inc.
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The Journal of Immunology: 205 (8)
The Journal of Immunology
Vol. 205, Issue 8
15 Oct 2020
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p53 Hinders CRISPR/Cas9-Mediated Targeted Gene Disruption in Memory CD8 T Cells In Vivo
Samarchith P. Kurup, Steven J. Moioffer, Lecia L. Pewe, John T. Harty
The Journal of Immunology October 15, 2020, 205 (8) 2222-2230; DOI: 10.4049/jimmunol.2000654

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p53 Hinders CRISPR/Cas9-Mediated Targeted Gene Disruption in Memory CD8 T Cells In Vivo
Samarchith P. Kurup, Steven J. Moioffer, Lecia L. Pewe, John T. Harty
The Journal of Immunology October 15, 2020, 205 (8) 2222-2230; DOI: 10.4049/jimmunol.2000654
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