Highly efficient genome editing via CRISPR-Cas9 ribonucleoprotein (RNP) delivery in mesenchymal stem cells
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초록
The CRISPR-Cas9 system has significantly advanced regenerativemedicine research by enabling genome editing in stemcells. Due to their desirable properties, mesenchymal stem cells(MSCs) have recently emerged as highly promising therapeuticagents, which properties include differentiation ability and cytokineproduction. While CRISPR-Cas9 technology is applied todevelop MSC-based therapeutics, MSCs exhibit inefficient genomeediting, and susceptibility to plasmid DNA. In this study,we compared and optimized plasmid DNA and RNP approachesfor efficient genome engineering in MSCs. The RNP-mediatedapproach enabled genome editing with high indel frequencyand low cytotoxicity in MSCs. By utilizing Cas9 RNPs, we successfullygenerated B2M-knockout MSCs, which reduced T-celldifferentiation, and improved MSC survival. Furthermore, thisapproach enhanced the immunomodulatory effect of IFN-r priming. These findings indicate that the RNP-mediated engineeringof MSC genomes can achieve high efficiency, and engineeredMSCs offer potential as a promising therapeutic strategy.
Abstract
The CRISPR-Cas9 system has significantly advanced regenerativemedicine research by enabling genome editing in stemcells. Due to their desirable properties, mesenchymal stem cells(MSCs) have recently emerged as highly promising therapeuticagents, which properties include differentiation ability and cytokineproduction. While CRISPR-Cas9 technology is applied todevelop MSC-based therapeutics, MSCs exhibit inefficient genomeediting, and susceptibility to plasmid DNA. In this study,we compared and optimized plasmid DNA and RNP approachesfor efficient genome engineering in MSCs. The RNP-mediatedapproach enabled genome editing with high indel frequencyand low cytotoxicity in MSCs. By utilizing Cas9 RNPs, we successfullygenerated B2M-knockout MSCs, which reduced T-celldifferentiation, and improved MSC survival. Furthermore, thisapproach enhanced the immunomodulatory effect of IFN-r priming. These findings indicate that the RNP-mediated engineeringof MSC genomes can achieve high efficiency, and engineeredMSCs offer potential as a promising therapeutic strategy.
참고문헌 (37)
- Mali P, "RNA-guided human genome engineering via Cas9", Science, 339, pp.823~826 (2013)
- Cong L, "Multiplex genome engineering using CRISPR", Cas systems, 2013, pp.339, 819~823
- Xiao A, "Chromosomal deletions and inversions mediated by TALENs and CRISPR", Cas in zebrafish, 2013, pp.41, e141~
- Hsu PD, "Development and applications of CRISPR-Cas9 for genome engineering", Cell, 157, pp.1262~1278 (2014)
- Ma X, "Small molecules promote CRISPR-Cpf1-mediated genome editing in human pluripotent stem cells", Nat Commun, 9, pp.1303~ (2018)
- DeWitt MA, "Selection-free genome editing of the sickle mutation in human adult hematopoietic stem", progenitor cells, 2016, pp.8, 360~
- Zhang Z, "CRISPR", Cas9genome-editing system in human stem cells : current status and future prospects, 2017, pp.9, 230~241
- Cox DB, "Therapeutic genome editing : prospects and challenges", Nat Med, 21, pp.121~131 (2015)
- Kim S, "Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins", Genome Res, 24, pp.1012~1019 (2014)
- Liang X, "Rapid and highly efficient mammalian cell engineering via Cas9 protein transfection", J Biotechnol, 208, pp.44~53 (2015)
- Cho SW, "Targeted genome engineering in human cells with the Cas9 RNAguided endonuclease", Nat Biotechnol, 31, pp.230~232 (2013)
- Pittenger MF, "Mesenchymal stem cell perspective : cell biology to clinical progress", NPJ Regen Med, 4, pp.22~ (2019)
- Reinders ME, "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, pp.107~111 (2013)
- Le Blanc K, "Mesenchymal stem cells for treatment of steroid-resistant, severe, acute graft-versus-host disease : a phase II study", Lancet, 371, pp.1579~1586 (2008)
- Cortez-Toledo E, "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, pp.114~119 (2019)
- Zhao K, "The clinical application of mesenchymal stromal cells in hematopoietic stem cell transplantation", J Hematol Oncol, 9, pp.46~ (2016)
- Le Blanc K, "HLA expression and immunologic properties of differentiated and undifferentiated mesenchymal stem cells", Exp Hematol, 31, pp.890~896 (2003)
- Ankrum JA, "Mesenchymal stem cells : immune evasive, not immune privileged", Nat Biotechnol, 32, pp.252~260 (2014)
- Park J, "Cas-Designer : a webbased tool for choice of CRISPR-Cas9 target sites", Bioinformatics, 31, pp.4014~4016 (2015)
- Lu Y, "Precise editing of a target base in the rice genome using a modified CRISPR", Cas9 system, 2017, pp.10, 523~525
- Lee BC, "Functional enhancement strategies for immunomodulation of mesenchymal stem cells and their therapeutic application", Stem Cell Res Ther, 11, pp.397~ (2020)
- Fu Y, "High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells", Nat Biotechnol, 31, pp.822~826 (2013)
- Schumann K, "Generation of knock-in primary human T cells using Cas9 ribonucleoproteins", Proc Natl Acad Sci U S A, 112, pp.10437~10442 (2015)
- Kim HJ, "Usage of human mesenchymal stem cells in cell-based therapy : advantages and disadvantages", Dev Reprod, 21, pp.1~10 (2017)
- Crippa S, "Mesenchymal stromal cells improve the transplantation outcome of CRISPR-Cas9 gene-edited human HSPCs", Mol Ther, 30, pp.3333~ (2022)
- Xu X, "Efficient homologydirected gene editing by CRISPR", Cas9 in human stem and primary cells using tube electroporation, 2018, pp.8, 11649~
- Fierro FA, "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, pp.1727~1737 (2011)
- Mancanares ACF, "Edition of prostaglandin E2 receptors EP2 and EP4 by CRISPR", Cas9 technology in equine adipose mesenchymal stem cells, 2020, pp.10, 1078~
- Lim J, "Small-sized mesenchymal stem cells with high glutathione dynamics show improved therapeutic potency in graft-versus-host disease", Clin Transl Med, 11, pp.e476~ (2021)
- Park DS, "Targeted base editing via RNA-Guided cytidine deaminases in Xenopus laevis embryos", Mol Cells, 40, pp.823~827 (2017)
- Richardson CD, "Enhancing homology-directed genome editing by catalytically active and inactive CRISPR-Cas9 using asymmetric donor DNA", Nat Biotechnol, 34, pp.339~344 (2016)
- Kweon J, "CRISPR", Cas-based customization of pooled CRISPR libraries, 2018, pp.13, e0199473~
- Kweon J, "A CRISPR-based base-editing screen for the functional assessment of BRCA1variants", Oncogene, 39, pp.30~35 (2020)
- Kim D, "Genome-wide target specificity of CRISPR RNA-guided adenine base editors", Nat Biotechnol, 37, pp.430~435 (2019)
- Kim D, "Digenome-seq : genomewide profiling of CRISPR-Cas9 off-target effects in human cells", Nat Methods, 12, pp.237~243 (2015)
- Bae S, "Cas-OFFinder : a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases", Bioinformatics, 30, pp.1473~1475 (2014)
- Park J, "Digenome-seq web tool for profiling CRISPR specificity", Nat Methods, 14, pp.548~549 (2017)