Highly efficient genome editing via CRISPR-Cas9 ribonucleoprotein (RNP) delivery in mesenchymal stem cells

A Reum Han; Ha Rim Shin; Jiyeon Kwon; Soo Been Lee; Sang Eun Lee; Eun-Young Kim; Jiyeon Kweon; Eun-Ju Chang; Yongsub Kim; Seong Who Kim

doi:10.5483/BMBRep.2023-0113

Highly efficient genome editing via CRISPR-Cas9 ribonucleoprotein (RNP) delivery in mesenchymal stem cells

BMB Rep. 2024 Jan;57(1):60-65. doi: 10.5483/BMBRep.2023-0113.

Authors

A Reum Han¹, Ha Rim Shin², Jiyeon Kwon³, Soo Been Lee⁴, Sang Eun Lee⁴, Eun-Young Kim⁴, Jiyeon Kweon³, Eun-Ju Chang⁴, Yongsub Kim⁵, Seong Who Kim⁶

Affiliations

¹ Department of Translational Medicine and Department of Biochemistry and Molecular Biology, Asan Medical Institute of Convergence Science and Technology, Asan Medical Center, University of Ulsan College of Medicine, Seoul 05505; Stem Cell Immunomodulation Research Center, University of Ulsan College of Medicine, Seoul 05505, Korea.
² Department of Cell and Genetic Engineering, Asan Medical Institute of Convergence Science and Technology, Asan Medical Center, University of Ulsan College of Medicine, Seoul 05505; Center for Genomic Integrity, Institute for Basic Science, Ulsan 44919; Stem Cell Immunomodulation Research Center, University of Ulsan College of Medicine, Seoul 05505, Korea.
³ Department of Cell and Genetic Engineering, Asan Medical Institute of Convergence Science and Technology, Asan Medical Center, University of Ulsan College of Medicine, Seoul 05505, Korea.
⁴ Department of Biochemistry and Molecular Biology, Asan Medical Institute of Convergence Science and Technology, Asan Medical Center, University of Ulsan College of Medicine, Seoul 05505; Stem Cell Immunomodulation Research Center, University of Ulsan College of Medicine, Seoul 05505, Korea.
⁵ Department of Cell and Genetic Engineering, Asan Medical Institute of Convergence Science and Technology, Asan Medical Center, University of Ulsan College of Medicine, Seoul 05505; Stem Cell Immunomodulation Research Center, University of Ulsan College of Medicine, Seoul 05505; Center for Cell therapy, Asan Medical Institute of Convergence Science and Technology, Asan Medical Center, Seoul 05505, Korea.
⁶ Department of Biochemistry and Molecular Biology Asan Medical Institute of Convergence Science and Technology, Asan Medical Center, University of Ulsan College of Medicine, Seoul 05505; Stem Cell Immunomodulation Research Center, University of Ulsan College of Medicine, Seoul 05505; Center for Cell therapy, Asan Medical Institute of Convergence Science and Technology, Asan Medical Center, Seoul 05505, Korea.

Abstract

The CRISPR-Cas9 system has significantly advanced regenerative medicine research by enabling genome editing in stem cells. Due to their desirable properties, mesenchymal stem cells (MSCs) have recently emerged as highly promising therapeutic agents, which properties include differentiation ability and cytokine production. While CRISPR-Cas9 technology is applied to develop MSC-based therapeutics, MSCs exhibit inefficient genome editing, and susceptibility to plasmid DNA. In this study, we compared and optimized plasmid DNA and RNP approaches for efficient genome engineering in MSCs. The RNP-mediated approach enabled genome editing with high indel frequency and low cytotoxicity in MSCs. By utilizing Cas9 RNPs, we successfully generated B2M-knockout MSCs, which reduced T-cell differentiation, and improved MSC survival. Furthermore, this approach enhanced the immunomodulatory effect of IFN-r priming. These findings indicate that the RNP-mediated engineering of MSC genomes can achieve high efficiency, and engineered MSCs offer potential as a promising therapeutic strategy. [BMB Reports 2024; 57(1): 60-65].

Publication types

News

MeSH terms

CRISPR-Cas Systems / genetics
DNA
Gene Editing*
Mesenchymal Stem Cells* / metabolism
Ribonucleoproteins / genetics
Ribonucleoproteins / metabolism

Substances

Ribonucleoproteins
DNA

Grants and funding

ACKNOWLEDGEMENTS This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (RS-2023-00260462, 2018R1A5A2020732, 2021R1C1C1007162, and 2023R1A2C2004057).