Elimination of Beta 2 Microglobulin Expression in HEK293 Cell Line Toward Producing HLA Class 1 Deficient Universal Cells

Background and Aim: In cell therapy, transplantation of allogeneichematopoietic stem cell (HSC) seems to be a beneficial and highthroughput method for not only hematologic but also non-hematologicand immunologic disorders. However, the use of HSC has somelimitations like; transplant rejection resulting from HLA mismatching between donor and recipient and identification of a suitable donor dueto the HLA polymorphism. Elimination of HLA class 1 from surfacesof HSCs by CRISPR/Cas9 system, that is easy to use the method withhigh efficiency compared to TALEN and ZFN, can be a way to reducetransplant rejection.Methods: In this study, we transfected dual gRNAs into HEK293T cellline using lipofectamine 2000 in order to a knockout B2M gene, whichis non-polymorphic and codes B2M protein which is essential for surfaceexpression of HLA class 1. Forty-eight hours after transfection, transfectedcells were separated by FACS. Due to the diversity of cell population,clonal single cell lines were isolated. DNA extraction followed by PCRand Sanger sequencing to detect the deletion in the targeted locus.Results: Two gRNAs were designed in this study, one for exon 1 and theother one for intron 1 to target the B2M gene. Using PCR, we observeddeletion of both alleles in B2M gene in 11.11% of sorted clonal celllines, and also only one allele of B2M gene was deleted in 22.22% of celllines. Sanger sequencing confirmed elimination of alleles.Conclusion: We revealed that a dual gRNAs can be a suitable approachto directly predicted deletions at any targeted loci and therefore can leadto a knockout gene. This process can also lead to generate a universal cellline for therapeutic aims in different patients regardless of HLA nature

کلیدواژه ها:

Transplant Rejection ، B2M ، HLA ، CRISPR/Cas9 ، Universal Cell