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The determination of null- or low-expressed HLA alleles is clinically relevant in both hematopoietic stem cell transplantation and solid organ transplantation. We studied the expression level of a questionable (Q)  HLA-B*38:68Q  allele, which carries a 9-nucleotide (nt) deletion at codon 230–232 in exon 4 of  HLA-B*38:01:01:01  using CRISPR/Cas9 gene editing technology. CRISPR/Cas9 gene editing of  HLA-B*38:01:01:01  homozygous EBV B cell line resulted in one  HLA-B*38:68Q/B*38:01:01:01  heterozygous and one  HLA-B*38:68Q  homozygous clone. Flow cytometric analysis of monoclonal anti-Bw4 antibody showed the protein expression of  HLA-B*38:01:01:01  in homozygous cells was 2.2 fold higher than  HLA-B*38:68Q/B*38:01:01:01  heterozygous cells, and the expression of  HLA-B*38:68Q/B*38:01:01:01  heterozygous cells was over 2.0 fold higher than  HLA-B*38:68Q  homozygous cells. The  HLA-B*38:68Q  expression was further confirmed using anti-B38 polyclonal antibody. Similarly, the expression of the  HLA-B*38:01:01:01  homozygous cells was 1.5 fold higher than that of  HLA-B*38:68Q/B*38:01:01:01  heterozygous cells, and the  HLA-B*38:68Q/B*38:01:01:01  heterozygous cells was over 1.6 fold higher than that of  HLA-B*38:68Q  homozygous cells. The treatment of  HLA-B*38:68Q  homozygous cells with IFN-γ significantly increased its expression. In conclusion, we demonstrate that  HLA-B*38:68Q  is a low-expressing HLA allele. The CRISPR/Cas9 technology is a useful tool to induce precise gene editing in HLA genes to enable the characterization of HLA gene variants on expression and function.

Integrate CRISPR/Cas9 for protein expression of HLA-B*38:68Q via precise gene editing