Effect of PD-1 knockout by CRISPR/Cas9 system on proliferation and IFN-γ secretion in human T lymphocytes
10.3872/j.issn.1007-385x.2019.06.007
- VernacularTitle:应用 CRISPR/Cas9 系统敲除 PD-1 基因对人 T 淋巴细胞增殖及分泌 IFN-γ的影响
- Author:
GONG Fusheng
;
XU Yangmei
;
LIU Shijia
;
HUANG Lijie
;
ZHENG Qiuhong
- Publication Type:Journal Article
- Keywords:
CRISPR/Cas9 system;
T lymphocyte;
nuclear transfection;
PD-1;
gene knockout;
IFN-γ
- From:
Chinese Journal of Cancer Biotherapy
2019;26(6):656-661
- CountryChina
- Language:Chinese
-
Abstract:
Objective: : To explore the effect of PD-1 gene knockout by CRISPR/Cas9 system on the proliferation and IFN-γ secretion in human T cells. Methods: : The sequence of sgRNA targeting PD-1 was designed. The PD-1-sgRNA and Cas9 mRNA were synthesized by T7 RNApolymerase in vitro, and then the mixture of PD-1-sgRNAand Cas9 mRNAwas delivered into activated T cells by nucleofection. The efficiency of gene knockout was confirmed by sequencing. The phenotypes of T lymphocytes and the expression of PD-1 after gene knockout were analyzed by Flow cytometry. The proliferation of T lymphocytes was calculated by trypan blue counting. The level of IFN-γ secreted by T lymphocytes was detected by ELISA. Results： ：PD-1-sgRNA and Cas9 mRNA were successfully synthesized in vitro and delivered into T cells by nucleofection. Sequencing technology confirmed that the PD-1 gene sequence was edited and the editing efficiency was 58.3%. The expression of PD-1 on T lymphocyte surface was down-regulated successfully by CRISPR/Cas9 system [(9.6±1.85)% vs (16.2±2.05)%, P<0.05]. The knockout of PD-1 gene did not affect the proliferation and phenotype of T lymphocytes(P<0.05); However, compared with the control group, the level of IFN-γ secreted by T lymphocytes in the PD-1sgRNA group was significantly increased (P<0.01). Conclusion: : CRISPR/Cas9 system can successfully ablate PD-1 gene in human T lymphocytes, which could block the negative regulation of PD-1／PD-L1 and further promote the IFN-γ secretion in T cells.
- Full text:20190607.pdf
