| Highly efficient genome editing via CRISPR-Cas9 ribonucleoprotein (RNP) delivery in mesenchymal stem cells |
Yongsub Kim  2,3,4,* (Associate Professor), Seong Who Kim1,3,4 (Professor), A.Reum Han1,3,# (Graduate student), Ha Rim Shin2,3,# (Graduate student), Jiyeon Kweon2,3 (Research Professor), Soo Been Lee1,3 (Graduate student), Sang Eun Lee1,3 (Research worker), Eun-Young Kim1,3 (Research Professor), Eun-Ju Chang1,3 (Professor) |
1Department of Biochemistry and Molecular Biology and 2Department of Cell and Genetic Engineering, Asan Medical Institute of Convergence Science and Technology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Republic of Korea,
3Stem Cell Immunomodulation Research Center, University of Ulsan College of Medicine, Seoul 05505, Republic of Korea,
4Center for Cell therapy, Asan Medical Institute of Convergence Science and Technology, Asan Medical Center, Seoul 05505, Republic of Korea |
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Abstract
The CRISPR-Cas9 system has significantly advanced regenerative medicine research by enabling genome editing in stem cells. Mesenchymal stem cells (MSCs) have recently emerged as highly promising therapeutic agents due to their desirable properties including differentiation ability and cytokine production. While CRISPR-Cas9 technology is being 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 RNP-mediated engineering of MSC genomes can achieve high efficiency, and engineered MSCs can potentially be a promising therapeutic strategy.
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| Abstract, Accepted Manuscript(in press) [Submitted on June 29, 2023, Accepted on August 31, 2023] |
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