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

Allogeneic stem cells have been introduced as a potential approach to generate grafts in regenerative medicine. But the clinical usage of them is limited due to the risk of immune rejection that is induced by the incompatibility of human leukocyte antigens (HLAs) between donors and recipients. To overcome this limitation, we knocked out the β2 microglobulin (B2M) gene which is crucial for HLA class I expression, using CRISPR/Cas9 approach. Non-viral transfer of two gRNAs targeting exon 1 and intron 1 in B2M gene caused in large and predictable deletion in region between two loci, which was defined by sequencing and polymerase chain reaction (PCR). Furthermore, results revealed that roughly 11.11% and 22.22% of the GFP expressing cells reflecting a homozygous and heterozygous pattern of genomic modifications. We demonstrated that the dual guide RNA approach is a simple and efficient method for gene disruption. Significantly, these engineered hypoimmunogenic cells could be proposed as universal cells that are not distinguishable to the recipient immune system in cell therapy and transplantation.