CRISPR-mediated downregulation of PD-1 expression on T cells

Abstract

Objective To investigate the feasibility of using clustered regularly interspaced short palindromic repeats (CRISPR)-mediated genome editing to downregulate the expression of programmed cell death protein 1 (PD-1) on primary T cells by using a lentivirus delivery system. Methods Lentivirus vectors pLentiCRISPR A1-A6 containing different PD-1 genomic DNA sequences as single guide RNA (sgRNA) for Cas9 targeting were constructed individually. The lentivirus vectors were tranduced into primary CD4 T cells. Flow cytometry analysis was performed to detect the expression of PD-1 for evaluating the knockout efficiency. Results The lentivirus vectors pLentiCRISPR A1-A6 carrying six different target sites were constructed and respectively tranduced into primary CD4 T cells. The expression of PD-1 accompanied with the activation of T cells. Co-expression of CD25 and PD-1 was observed on activated T cells. All of the six sites could be targeted by Cas9, of which A2 and A6 sites were more efficient in knocking out the gene encoding PD-1 with a rate of 19% and 29%, respectively. Conclusion This study suggests that it is feasible to knock out the expression of PD-1 on primary T cells by using CRISPR. Key words: CRISPR; PD-1; CD25