BMB Reports

Korean Society for Biochemistry and Molecular Biology (생화학분자생물학회)
권호 표지
DOI QR코드

DOI QR Code

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

  • A Reum Han (Department of Translational Medicine, Asan Medical Institute of Convergence Science and Technology, Asan Medical Center, University of Ulsan College of Medicine) ;
  • Ha Rim Shin (Department of Cell and Genetic Engineering, Asan Medical Institute of Convergence Science and Technology, Asan Medical Center, University of Ulsan College of Medicine) ;
  • Jiyeon Kweon (Department of Cell and Genetic Engineering, Asan Medical Institute of Convergence Science and Technology, Asan Medical Center, University of Ulsan College of Medicine) ;
  • Soo Been Lee (Department of Biochemistry and Molecular Biology, Asan Medical Institute of Convergence Science and Technology, Asan Medical Center, University of Ulsan College of Medicine) ;
  • Sang Eun Lee (Department of Biochemistry and Molecular Biology, Asan Medical Institute of Convergence Science and Technology, Asan Medical Center, University of Ulsan College of Medicine) ;
  • Eun-Young Kim (Department of Biochemistry and Molecular Biology, Asan Medical Institute of Convergence Science and Technology, Asan Medical Center, University of Ulsan College of Medicine) ;
  • Jiyeon Kweon (Department of Cell and Genetic Engineering, Asan Medical Institute of Convergence Science and Technology, Asan Medical Center, University of Ulsan College of Medicine) ;
  • Eun-Ju Chang (Department of Biochemistry and Molecular Biology, Asan Medical Institute of Convergence Science and Technology, Asan Medical Center, University of Ulsan College of Medicine) ;
  • Yongsub Kim (Department of Cell and Genetic Engineering, Asan Medical Institute of Convergence Science and Technology, Asan Medical Center, University of Ulsan College of Medicine) ;
  • Seong Who Kim (Department of Biochemistry and Molecular Biology, Asan Medical Institute of Convergence Science and Technology, Asan Medical Center, University of Ulsan College of Medicine)
  • Received : 2023.06.29
  • Accepted : 2023.08.31
  • Published : 2024.01.31
Download PDF
⟨ Previous Next ⟩

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.

Keywords

Acknowledgement

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).

References

  1. Mali P, Yang L, Esvelt KM et al (2013) RNA-guided human genome engineering via Cas9. Science 339, 823-826 https://doi.org/10.1126/science.1232033
  2. Cong L, Ran FA, Cox D et al (2013) Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819-823 https://doi.org/10.1126/science.1231143
  3. Xiao A, Wang Z, Hu Y et al (2013) Chromosomal deletions and inversions mediated by TALENs and CRISPR/Cas in zebrafish. Nucleic Acids Res 41, e141
  4. Hsu PD, Lander ES and Zhang F (2014) Development and applications of CRISPR-Cas9 for genome engineering. Cell 157, 1262-1278 https://doi.org/10.1016/j.cell.2014.05.010
  5. Ma X, Chen X, Jin Y et al (2018) Small molecules promote CRISPR-Cpf1-mediated genome editing in human pluripotent stem cells. Nat Commun 9, 1303
  6. DeWitt MA, Magis W, Bray NL et al (2016) Selection-free genome editing of the sickle mutation in human adult hematopoietic stem/progenitor cells. Sci Transl Med 8, 360ra134
  7. Zhang Z, Zhang Y, Gao F et al (2017) CRISPR/Cas9 genome-editing system in human stem cells: current status and future prospects. Mol Ther Nucleic Acids 9, 230-241 https://doi.org/10.1016/j.omtn.2017.09.009
  8. Cox DB, Platt RJ and Zhang F (2015) Therapeutic genome editing: prospects and challenges. Nat Med 21, 121-131 https://doi.org/10.1038/nm.3793
  9. Kim S, Kim D, Cho SW, Kim J and Kim JS (2014) Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins. Genome Res 24, 1012-1019 https://doi.org/10.1101/gr.171322.113
  10. Liang X, Potter J, Kumar S et al (2015) Rapid and highly efficient mammalian cell engineering via Cas9 protein transfection. J Biotechnol 208, 44-53 https://doi.org/10.1016/j.jbiotec.2015.04.024
  11. Cho SW, Kim S, Kim JM and Kim JS (2013) Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease. Nat Biotechnol 31, 230-232 https://doi.org/10.1038/nbt.2507
  12. Pittenger MF, Discher DE, Peault BM, Phinney DG, Hare JM and Caplan AI (2019) Mesenchymal stem cell perspective: cell biology to clinical progress. NPJ Regen Med 4, 22
  13. Reinders ME, de Fijter JW, Roelofs H et al (2013) Autologous bone marrow-derived mesenchymal stromal cells for the treatment of allograft rejection after renal transplantation: results of a phase I study. Stem Cells Transl Med 2, 107-111 https://doi.org/10.5966/sctm.2012-0114
  14. Le Blanc K, Frassoni F, Ball L et al (2008) Mesenchymal stem cells for treatment of steroid-resistant, severe, acute graft-versus-host disease: a phase II study. Lancet 371, 1579-1586 https://doi.org/10.1016/S0140-6736(08)60690-X
  15. Cortez-Toledo E, Rose M, Agu E et al (2019) Enhancing Retention of human bone marrow mesenchymal stem cells with prosurvival factors promotes angiogenesis in a mouse model of limb ischemia. Stem Cells Dev 28, 114-119 https://doi.org/10.1089/scd.2018.0090
  16. Zhao K and Liu Q (2016) The clinical application of mesenchymal stromal cells in hematopoietic stem cell transplantation. J Hematol Oncol 9, 46
  17. Le Blanc K, Tammik C, Rosendahl K, Zetterberg E and Ringden O (2003) HLA expression and immunologic properties of differentiated and undifferentiated mesenchymal stem cells. Exp Hematol 31, 890-896 https://doi.org/10.1016/S0301-472X(03)00110-3
  18. Ankrum JA, Ong JF and Karp JM (2014) Mesenchymal stem cells: immune evasive, not immune privileged. Nat Biotechnol 32, 252-260
  19. Park J, Bae S and Kim JS (2015) Cas-Designer: a web-based tool for choice of CRISPR-Cas9 target sites. Bioinformatics 31, 4014-4016 https://doi.org/10.1093/bioinformatics/btv537
  20. Lu Y and Zhu JK (2017) Precise editing of a target base in the rice genome using a modified CRISPR/Cas9 system. Mol Plant 10, 523-525 https://doi.org/10.1016/j.molp.2016.11.013
  21. Lee BC and Kang KS (2020) Functional enhancement strategies for immunomodulation of mesenchymal stem cells and their therapeutic application. Stem Cell Res Ther 11, 397
  22. Fu Y, Foden JA, Khayter C et al (2013) High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat Biotechnol 31, 822-826 https://doi.org/10.1038/nbt.2623
  23. Schumann K, Lin S, Boyer E et al (2015) Generation of knock-in primary human T cells using Cas9 ribonucleoproteins. Proc Natl Acad Sci U S A 112, 10437-10442 https://doi.org/10.1073/pnas.1512503112
  24. Kim HJ and Park JS (2017) Usage of human mesenchymal stem cells in cell-based therapy: advantages and disadvantages. Dev Reprod 21, 1-10 https://doi.org/10.12717/DR.2017.21.1.001
  25. Crippa S, Conti A, Vavassori V et al (2022) Mesenchymal stromal cells improve the transplantation outcome of CRISPR-Cas9 gene-edited human HSPCs. Mol Ther 30, 3333
  26. Xu X, Gao D, Wang P et al (2018) Efficient homology-directed gene editing by CRISPR/Cas9 in human stem and primary cells using tube electroporation. Sci Rep 8, 11649
  27. Fierro FA, Kalomoiris S, Sondergaard CS and Nolta JA (2011) Effects on proliferation and differentiation of multipotent bone marrow stromal cells engineered to express growth factors for combined cell and gene therapy. Stem Cells 29, 1727-1737 https://doi.org/10.1002/stem.720
  28. Mancanares ACF, Cabezas J, Manriquez J et al (2020) Edition of prostaglandin E2 receptors EP2 and EP4 by CRISPR/Cas9 technology in equine adipose mesenchymal stem cells. Animals (Basel) 10, 1078
  29. Lim J, Heo J, Yu HY et al (2021) Small-sized mesenchymal stem cells with high glutathione dynamics show improved therapeutic potency in graft-versus-host disease. Clin Transl Med 11, e476
  30. Park DS, Yoon M, Kweon J, Jang AH, Kim Y and Choi SC (2017) Targeted base editing via RNA-Guided cytidine deaminases in Xenopus laevis embryos. Mol Cells 40, 823-827
  31. Richardson CD, Ray GJ, DeWitt MA, Curie GL and Corn JE (2016) Enhancing homology-directed genome editing by catalytically active and inactive CRISPR-Cas9 using asymmetric donor DNA. Nat Biotechnol 34, 339-344 https://doi.org/10.1038/nbt.3481
  32. Kweon J, Kim DE, Jang AH and Kim Y (2018) CRISPR/Cas-based customization of pooled CRISPR libraries. PLoS One 13, e0199473
  33. Kweon J, Jang AH, Shin HR et al (2020) A CRISPR-based base-editing screen for the functional assessment of BRCA1 variants. Oncogene 39, 30-35 https://doi.org/10.1038/s41388-019-0968-2
  34. Kim D, Kim DE, Lee G, Cho SI and Kim JS (2019) Genome-wide target specificity of CRISPR RNA-guided adenine base editors. Nat Biotechnol 37, 430-435 https://doi.org/10.1038/s41587-019-0050-1
  35. Kim D, Bae S, Park J et al (2015) Digenome-seq: genome-wide profiling of CRISPR-Cas9 off-target effects in human cells. Nat Methods 12, 237-243 https://doi.org/10.1038/nmeth.3284
  36. Bae S, Park J and Kim JS (2014) Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases. Bioinformatics 30, 1473-1475 https://doi.org/10.1093/bioinformatics/btu048
  37. Park J, Childs L, Kim D et al (2017) Digenome-seq web tool for profiling CRISPR specificity. Nat Methods 14, 548-549 https://doi.org/10.1038/nmeth.4262