Preparation of B2M and PD-1 Efficiently Double Genes Knock-out Primary Human T Lymphocytes by Cas9 RNP Technology

Objective To observe the efficiency of Cas9 RNP technology for the preparation of B2M and PD-1 double genes knock-out primary human T lymphocytes (hereinafter referred to as T cells).

Methods Single-guide RNA (sgRNA) was designed and cloned into vector plasmids. The effective sgRNAs were screened out by Sanger sequencing in HEK293T cells. B2M/PD-1 single and double genes knock-out T cells were constructed respectively and successively. The editing efficiencies were verified by Sanger sequencing, TA cloning and flow cytometry.

Results The vector plasmids containing sgRNAs were successfully constructed and the high-efficiency sgRNAs (B2M sgRNA1 and PD-1 sgRNA1) were obtained. The editing efficiencies of B2M/PD-1 single gene knocked-out T cells were both up to 90%. In double genes knock-out T cells, the editing efficiencies at gene level were up to 90%, while the down-regulation rates of B2M and PD-1 protein reached 86% and 89%, respectively.

Conclusion Cas9 RNP technology could be used to prepare B2M and PD-1 efficiently double genes knock-out T cells.