CRISPR/Cas9 Ribonucleoprotein Complex-Mediated Efficient B2M Knockout in Human Induced Pluripotent Stem Cells (iPSCs)
Issued Date
2022-01-01
Resource Type
ISSN
10643745
eISSN
19406029
Scopus ID
2-s2.0-85132455030
Pubmed ID
33945142
Journal Title
Methods in Molecular Biology
Volume
2454
Start Page
607
End Page
624
Rights Holder(s)
SCOPUS
Bibliographic Citation
Methods in Molecular Biology Vol.2454 (2022) , 607-624
Suggested Citation
Thongsin N., Wattanapanitch M. CRISPR/Cas9 Ribonucleoprotein Complex-Mediated Efficient B2M Knockout in Human Induced Pluripotent Stem Cells (iPSCs). Methods in Molecular Biology Vol.2454 (2022) , 607-624. 624. doi:10.1007/7651_2021_352 Retrieved from: https://repository.li.mahidol.ac.th/handle/20.500.14594/83902
Title
CRISPR/Cas9 Ribonucleoprotein Complex-Mediated Efficient B2M Knockout in Human Induced Pluripotent Stem Cells (iPSCs)
Author(s)
Author's Affiliation
Other Contributor(s)
Abstract
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.