B2M gene knockout in HEK293T cells by non-viral delivery of CRISPR-Cas9 system for the generation of universal cells

Abstract

Background: Allogeneic stem cells are the most potent sources for replacing cell, tissue, and organ malfunctions. The clinical use of these stem cells has been limited due to the risk of immune system rejection due to the incompatibility of human leukocyte (HLA) antigens between donors and recipients. To overcome this limitation, we used the CRISPR/Cas9 system to eliminate the β2 microglobulin (B2M) gene, which plays a vital role in the expression of HLA class I. Results: Non-viral transfer of two gRNAs targeting the first exon and intron in the B2M gene results in large deletions in the target region. In addition, the results of this study showed that 11.11% and 22.22% of cells received genomic changes as homozygous and heterozygous, respectively. Conclusion: In conclusion, we have shown that the dual guide RNA strategy is a simple and efficient method for modifying genes. As a result, these cells can be proposed as universal cells that are not detectable in the cell therapy system and transplantation by the receptor immune system.