CRISPR/Cas9 Ribonucleoprotein Complex-Mediated Efficient B2M Knockout in Human Induced Pluripotent Stem Cells (iPSCs)

• Main Author: Thongsin N.
• Other Authors: Mahidol University
• Format: Book Chapter
• Published: 2023
• Subjects: Biochemistry, Genetics and Molecular Biology
• Online Access: https://repository.li.mahidol.ac.th/handle/123456789/83902

Advances in induced pluripotent stem cell (iPSC) technology provide a renewable source of cells for tissue regeneration and therefore hold great promise for cell replacement therapy. However, immune rejection of allograft due to human leukocyte antigen (HLA) mismatching remains a major challenge. Considerable efforts have been devoted to overcoming the immunogenicity of allograft transplantation. One of the approaches is an elimination of HLA molecules on the surface of allogeneic cells using genome editing technology to generate universal stem cells. Here, we present a simple and effective genome editing approach to knockout the β-2-immunoglobulin (B2M) gene, which encodes B2M protein that forms a heterodimer with HLA class I proteins, in induced pluripotent stem cells (iPSCs) leading to HLA class I (HLA-I) depletion. We also describe detailed procedures for validation of the B2M-knockout iPSCs using flow cytometry, and genotypic analysis for potential off-target regions. Our protocol is also applicable for knocking out other genes in iPSCs and other cell types.