Human leukocyte antigens (HLAs) are essential immune molecules that affect transplantation and adoptive immunotherapy. When hematopoietic stem cells or organs are transplanted with HLA-mismatched recipients， graft-versus-host disease or graft rejection can be induced by allogeneic immune responses. The function of each HLA allele has been studied using HLA-deficient cells generated from mutant cell lines or by RNA interference， zinc finger nuclease， and the CRISPR/Cas9 system. To improve HLA gene editing， we attempted to generate an HLA class I null cell line using the multiplex CRISPR/Cas9 system by targeting exons 2 and 3 of HLA-A， HLA-B， and HLA-C genes simultaneously. Multiplex HLA editing could induce the complete elimination of HLA class I genes by bi-allelic gene disruption on target sites which was defined by flow cytometry and target-specific polymerase chain reaction. Furthermore， artificial antigen-presenting cells were generated by transfer of a single HLA class I allele and co-stimulatory molecules into this novel HLA class I null cell line. Artificial antigen-presenting cells showed HLA-restricted antigen presentation following antigen processing and were successfully used for the efficient generation of tumor antigen-specific cytotoxic T cells in vitro. The efficient editing of HLA genes may provide a basis for universal cellular therapies and transplantation.