Department of Cellular Biology, University of Georgia, Athens, GA 30602.
Center for Tropical and Emerging Global Diseases, University of Georgia, Athens, GA 30602.
J Immunol. 2020 Oct 15;205(8):2222-2230. doi: 10.4049/jimmunol.2000654. Epub 2020 Sep 4.
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.
CRISPR/Cas9 技术彻底改变了细胞内快速可靠的基因编辑。尽管许多细胞类型都经历了 CRISPR/Cas9 介导的基因编辑，但在 Ag 经验记忆 CD8 T 细胞的遗传改变方面尚无成功证据。在这项研究中，我们表明，CRISPR/Cas9 介导的记忆 CD8 T 细胞中的基因编辑会阻止它们在体内再次遇到 Ag 后的增殖。这种缺陷是由促凋亡转录因子 p53 介导的，p53 是 DNA 损伤的传感器。暂时抑制 p53 功能为记忆 CD8 T 细胞提供了一个修复 DNA 损伤的机会，有利于在再次遇到 Ag 时产生强大的回忆反应。我们通过在小鼠模型中使用 p53siRNA 下的 CRISPR/Cas9 介导的靶向基因敲除来改变记忆 CD8 T 细胞的功能，从而证明了这一点。我们的方法因此适应了 CRISPR/Cas9 技术，使记忆 CD8 T 细胞能够首次在体内进行基因编辑，据我们所知。
