[摘要] 目的：探讨应用CRISPR/Cas9 基因编辑系统敲除PD-1 基因对人T 细胞增殖及分泌IFN-γ 的影响。方法：设计靶向PD-1 基因的sgRNA 序列，应用T7 RNA聚合酶体外分别合成PD-1-sgRNA 和Cas9mRNA。利用核转染技术将PD-1-sgRNA 和Cas9 mRNA混合物转入激活的人T淋巴细胞，测序确认基因敲除的效率。应用流式细胞术分析基因敲除后T淋巴细胞表型和PD-1 的表达情况,锥虫蓝活细胞计数法检测T 细胞增殖活力,ELISA 检测T 细胞分泌IFN-γ 情况。结果：体外成功合成PD-1-sgRNA和Cas9 mRNA。将PD-1-sgRNA和Cas9 mRNA核转染T淋巴细胞，测序证实PD-1 基因序列编辑效率为58.3%。CRISPR/Cas9 系统成功下调T淋巴细胞表面PD-1 分子的表达水平[ （9.6±1.85）% vs（16.2±2.05）%，P<0.05]，PD-1 基因敲除不影响T 细胞的增殖活力和细胞表型(P>0.05)；但PD-1-sgRNA 组的效应T细胞分泌IFN-γ 水平显著升高(P<0.01)。结论：CRISPR/Cas9 基因编辑系统成功敲除人T淋巴细胞PD-1 基因，降低PD-1 分子表达可阻断PD-1／PD-L1 负性调控从而增强T细胞的免疫活性，且促进效应T细胞分泌IFN-γ。

[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 RNA polymerase in vitro, and then the mixture of PD-1-sgRNA and Cas9 mRNA was 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-1-sgRNA 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.

福建省自然科学基金资助项目（No. 2017J01264）；福建省科技计划资助项目（No.2018Y2003）