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Archive > Volume 26 Issue 6 > 2019,26(6):656-661. DOI:10.3872/j.issn.1007-385X.2019.06.007 Prev Next
Basic Research
Effect of PD-1 knockout by CRISPR/Cas9 system on proliferation and IFN- γ secretion in human T lymphocytes
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Abstract:
[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.
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Project Supported:
Project supported by the Natural Science Foundation of Fujian Province (No. 2017J01264), and the Science and Technology Program of Fujian Province (No.2018Y2003)
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