A Comparative Study on the Efficiency of CRISPR-Cas9 in Human Embryonic Kidney 293 Cells and Peripheral Blood Mononuclear Cells for Disruption in Programmed Cell Death Protein 1

Abstract

CRISPR-Cas9 is the most important tool in genome engineering in recent years. The efficiency of this instrument on active and non-active genes is variable. Programmed cell death protein 1(PD-1) is a surface acceptor on T cells, B cells, and dendritic cells. This protein has an important role in the production of inducing tolerance in lymphocytes. Nowadays, this characteristic is used in cell therapy and immunotherapy of cancer. In the present study, the peripheral blood mononuclear cells and HEK293 cells were selected as expression and non-expression cells of the PD-1 gene. Six pairs of sgRNA were designed for the PD-1 gene. The transfected cells were sorted by the FACS machine. A common pair of primers were used for amplification of cute regions. Px458 was used as an expressional vector for the transfection of PBMCs and HEK293. Transfection was done using lipofectamine and electroporation methods. In PBMCs, 2 guides, sgRNA (3+1) and sgRNA (3+5) were able to disrupt the PD-1 gene. In contrast, in HEK293, none of the 6 guides were able to disrupt it. According to the results obtained, the PD-1 gene cutting in HEK293 cells was failed. However, it was successful in PBMCs. Therefore, it can be told that the heterochromatin region or other genome remodeling mechanisms such as epigenetic remodeling inhibit the PD-1 gene cutting by CRISPR-Cas9 in HEK293 cells.

Keywords

• crispr-cas9
• dual-transfection heterochromatin
• pbmcs
• pd-1 knockout