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.
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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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