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

Document Type : Research Article


1 Department of Genetics, Marvdasht Branch, Islamic Azad University, Marvdasht, Iran

2 Department of Medical Genetics, School of Medicine, Shiraz University of Medical Sciences, Shiraz Iran

3 Stem Cell Technology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran


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.


Alambeladi S, Hosseiny S, Jafarinia M, Dianatpour M. 2021. Use of dual-transfection for programmed cell death protein 1 disruption mediated by CRISPR-Cas9 in human peripheral blood mononuclear cells. Iran J Basic Med Sci 24(1):44-50.
Bally AP, Austin JW, Boss JM. 2016. Genetic and epigenetic regulation of PD-1 expression. J Immunol 196(6):2431-2437.
Blank C, Kuball J, Voelkl S, Wiendl H, Becker, B, Walter B, ... and Mackensen A. 2006. Blockade of PD-L1 (B7-H1) augments human tumor-specific T cell responses in vitro. Int J Cancer 119(2):317-327.
Cho SW, Kim S, Kim JM, Kim JS. 2013. Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease. Nat Biotechnol 31(3):230-232.
Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, ... & Zhang F. 2013. Multiplex genome engineering using CRISPR/Cas systems. Science 339(6121):819-823.
Cong L, Zhang F. Genome engineering using CRISPR-Cas9 system. 2015. Methods Mol Biol 1239:197‐217.
Hsu PD, Scott DA, Weinstein JA, Ran FA, Konermann S, Agarwala V, ... & Zhang, F.2013.  DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol 31(9):827-832.
John LB, Devaud C, Duong CP, Yong CS, Beavis PA, Haynes NM, ... & Darcy PK. 2013. Anti-PD-1 antibody therapy potently enhances the eradication of established tumors by gene-modified T cells. Clin Cancer Res 19(20):5636-5646.
Kallimasioti-Pazi EM, Thelakkad Chathoth K, Taylor GC, Meynert A, Ballinger T, Kelder MJ, ... & Wood AJ. 2018. Heterochromatin delays CRISPR-Cas9 mutagenesis but does not influence the outcome of mutagenic DNA repair. PLoS Biol 16(12): 2005595.
Keir ME, Butte MJ, Freeman GJ, Sharpe AH. 2008. PD-1 and its ligands in tolerance and immunity. Annu Rev Immunol 26:677-704.
Lioyd A, Vickery ON, Laugel B. 2013. Beyond the antigen receptor: editing the genome of T-cells for cancer adoptive cellular therapies, Front Immunol 5(4):221.
Luke JJ, Ott PA. 2015. PD-1 pathway inhibitors: the next generation of immunotherapy for advanced melanoma. Oncotarget 6(6):3479-3492.
Makarova KS, Haft DH, Barrangou R, Brouns S J, Charpentier E, Horvath P, ... & Koonin E V. 2011. Evolution and classification of the CRISPR-Cas systems. Nat Rev Microbiol 9(6):467-477.
Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, ... & Church GM. 2013. RNA-guided human genome engineering via Cas9. Science 339(6121):823-826.
Pen JJ, Keersmaecker BD, Heirman C, Corthals J, Liechtenstein T, Escors D, ... & Breckpot, K. 2014. Interference with PD-L1/PD-1 co-stimulation during antigen presentation enhances the multifunctionality of antigen-specific T cells. Gene Ther 21(3):262-271.
Qi LS, Larson MH, Gilbert LA, Doudna JA, Weissman JS, Arkin AP, Lim WA. 2013. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 152(5):1173-1183.
Ran FA, Hsu PD, Lin CY, Gootenberg JS, Konermann S, Trevino AE, ... & Zhang F. 2013. Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell 154(6):1380-1389.
Su S, Hu B, Shao J, Shen B, Du J, Du Y, ... & Liu B. 2016. CRISPR-Cas9 mediated efficient PD-1 disruption on human primary T cells from cancer patients. Sci Rep 286:20070.
Tumeh PC, Harview CL, Yearley JH, Shintaku I P, Taylor EJ, Robert L, ... & Ribas A. 2014. PD-1 blockade induces responses by inhibiting adaptive immune resistance. Nature 515(7528):568‐571.
Wang W, Lau R, Yu D, Zhu W, Korman A, Weber J. 2009. PD-1 blockade reverses the suppression of melanoma antigen-specific CTL by CD4+ CD25 (Hi) regulatory T cells. Int Immunol 21(9):1065-1077.