Use of dual-transfection for programmed death cell protein 1 disruption mediated by CRISPR-Cas9 in human peripheral blood mononuclear cells

Document Type : Original Article


1 Department of Genetics, Fars Science and Research Branch, Islamic Azad University, Marvdasht Iran

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

3 Department of Physiology, Shiraz Branch, Islamic Azad University, Shiraz Iran

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

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


Objective(s): Checkpoint blocking is considered a revolutionary method in cancer treatment. This method eliminates cancer cells by maintaining the sensitivity of immune cells. Today, cell therapy through checkpoint blocking is known as the most efficient method of cancer treatment. The programmed cell death protein-1(PD-1), as an immune check protein, has a vital role in weakening the immune responses by reducing the number of stimulated T cells. In normal situations, a decline in the immune responses can cause induced tolerance and prevent autoimmune diseases.
Materials and Methods: In this study, to reduce the induction of tolerance due to PDL-1 binding to PD-1, the PD-1 gene was destroyed in PBMCs by the means of CRISPR-Cas9 and dual-transfection of two plasmids containing the Cas 9 gene and two different sgRNAs specific to two region of PD-1 gene in order to produce a deletion mutation.  Six different sgRNA were designed and cloned in PX-458 plasmid vector, and PBMCs were transfected using lipofectamine 2000 and electroporation. Indels were evaluated by gel electrophoresis and Sanger sequencing.  
Results: We showed the PD-1 gene in PBMCs was knocked out successfully by CRISPR-Cas9 and dual-transfection of two sgRNAs. The minimum interval between the two sgRNAs was 448 nucleotides.
Conclusion: The results of this research demonstrated that the use of dual-transfection of CRISPR-Cas9 sgRNA is a suitable method to knock out the PD-1 gene and prevention of inducing tolerance in PBMCs.


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