Potential Applications of the CRISPR-Cas9 System for Research and Treatment of Osteoarthritis

Abstract

Osteoarthritis is a common degenerative disease of joint cartilage that affects millions of people in the world, especially the elderly. Progression of osteoarthritis is associated with a plethora of genetic and non-genetic factors. The CRISPR/Cas9 system is emerging as a powerful tool for genome engineering and has remarkable potential for guiding further research into osteoarthritis and may be a viable means for treating the disease. This review discusses existing and potential applications of the CRISPR/Cas9 system in osteoarthritis studies and treatments. Firstly, we briefly summarize the current status and mechanism of this technology. Next, we focus on the latest advances in the application of CRISPR/Cas9 system in elucidating the contributions of various factors to the pathogenesis of osteoarthritis as demonstrated through in vitro studies and animal models. Finally, we provide our perspective on the direction and challenges of studying and treating osteoarthritis with CRISPR/Cas9.

Zusammenfassung

Osteoarthrose ist eine weitverbreitete degenerative Erkrankung des Gelenkknorpels, von der weltweit Millionen Menschen – insbesondere ältere Erwachsene – betroffen sind. Die Krankheitsprogression steht mit einer Vielzahl genetischer und nicht genetischer Faktoren in Zusammenhang. Das CRISPR/Cas9-System etabliert sich als leistungsstarkes Werkzeug der Genom-Editierung, das ein bemerkenswertes Potenzial für die Erforschung der Osteoarthrose besitzt und möglicherweise als geeignete Methode zur Therapie der Erkrankung dienen könnte. Diese Übersichtsarbeit diskutiert bestehende und potenzielle Anwendungen des CRISPR/Cas9-Systems in der Osteoarthrose-Forschung und -Behandlung. Zunächst fassen wir kurz den aktuellen Stand und die Funktionsweise dieser Technologie zusammen. Anschließend konzentrieren wir uns auf die neuesten Fortschritte bei der Anwendung des CRISPR/Cas9-Systems zur Aufklärung des Beitrags verschiedener Faktoren zur Pathogenese der Osteoarthrose, wie sie in In-vitro-Studien und Tiermodellen demonstriert wurden. Abschließend geben wir einen Ausblick auf zukünftige Richtungen und Herausforderungen bei der Erforschung und Behandlung der Osteoarthrose mithilfe von CRISPR/Cas9.

Keywords

CRISPR/Cas9 - osteoarthritis - mechanism - treatment

Schlüsselwörter

CRISPR/Cas9 - Arthrose - Mechanismus - Behandlung

Publication History

Received: 07 January 2025

Accepted after revision: 16 May 2025

Article published online:
24 June 2025

• References

• 1 Cong L, Ran FA, Cox D. Multiplex genome engineering using CRISPR/Cas systems. Science 2013; 339: 819-823
• 2 Mali P, Yang L, Esvelt KM. et al. RNA-guided human genome engineering via Cas9. Science 2013; 339: 823-826
• 3 Jinek M, Chylinski K, Onfara I. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 2012; 337: 816-821
• 4 Ishino Y, Shinagawa H, Makino K. et al. Nucleotide sequence of the iap gene, responsible for alkaline phosphatase isozyme conversion in escherichia coli, and identification of the gene product. J Bacteriol 1987; 169: 5429-5433
• 5 Jansen R, Embden JD, Gaastra W. et al. Identification of genes that are associated with DNA repeats in prokaryotes. Mol Microbiol 2002; 43: 1565-1575
• 6 Mojica FJ, Díez-Villaseñor C, García-Martínez J. et al. Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements. J Mol Evol 2005; 60: 174-182
• 7 Deltcheva E, Chylinski K, Sharma CM. et al. CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature 2011; 471: 602-607
• 8 Ye X, Lin J, Chen Q. et al. An Efficient Vector-Based CRISPR/Cas9 System in Zebrafish Cell Line. Mar Biotechnol (NY) 2024; 26: 588-598
• 9 Lebek S, Caravia XM, Straub LG. et al. CRISPR-Cas9 base editing of pathogenic CaMKIIδ improves cardiac function in a humanized mouse model. J Clin Invest 2024; 134: e175164
• 10 Ge W, Gou S, Zhao X. et al. In vivo evaluation of guide-free Cas9-induced safety risks in a pig model. Signal Transduct Target Ther 2024; 9: 184
• 11 Ito Y, Inoue S, Nakashima T. et al. Epigenetic profiles guide improved CRISPR/Cas9-mediated gene knockout in human T cells. Nucleic Acids Res 2024; 52: 141-153
• 12 Baker M, Callaway E, Castelvecchi D. et al. A. 365 days: The science events that shaped 2015. Nature 2015; 528: 448-451
• 13 McNutt M. Breakthrough to genome editing. Science 2015; 350: 1445
• 14 Minnig MCC, Golightly YM, Nelson AE. Epidemiology of osteoarthritis: literature update 2022–2023. Curr Opin Rheumatol 2024; 36: 108-112
• 15 Zeng D, Umar M, Zhu Z. et al. Development of novel osteoarthritis therapy by targeting AMPK-β-catenin-Runx2 signaling. Genes Dis 2025; 12: 101247
• 16 Tang S, Zhang C, Oo WM. et al. Osteoarthritis. Nat Rev Dis Primers 2025; 11: 10
• 17 van Saase JL, van Romunde LK, Cats A. et al. Epidemiology of osteoarthritis: Zoetermeer survey. Comparison of radiological osteoarthritis in a Dutch population with that in 10 other populations. Ann Rheum Dis 1989; 48: 271-280
• 18 Glyn-Jones S, Palmer AJ, Agricola R. et al. Osteoarthritis. Lancet 2015; 386: 376-387
• 19 Cao F, Xu Z, Li XX. et al. Trends and cross-country inequalities in the global burden of osteoarthritis, 1990–2019: A population-based study. Ageing Res Rev 2024; 99: 102382
• 20 Yu Q, Xiao Y, Guan M. et al. Regulation of ferroptosis in osteoarthritis and osteoarthritic chondrocytes by typical MicroRNAs in chondrocytes. Front Med (Lausanne) 2024; 11: 1478153
• 21 Yokota S, Ishizu H, Miyazaki T. et al. Osteoporosis, Osteoarthritis, and Subchondral Insufficiency Fracture: Recent Insights. Biomedicines 2024; 12: 843
• 22 Deveza LA, Loeser RF. Is osteoarthritis one disease or a collection of many? Rheumatology (Oxford). 2018; 57 (Suppl. 4) iv34-iv42
• 23 Xia B, Di C, Zhang J. et al. Osteoarthritis pathogenesis: A review of molecular mechanisms. Calcif Tissue Int 2014; 95: 495-505
• 24 Grol MW. The evolving landscape of gene therapy strategies for the treatment of osteoarthritis. Osteoarthritis Cartilage 2024; 32: 372-384
• 25 Jiang F, Doudna JA. CRISPR-Cas9 structures and mechanisms. Ann Rev Biophys 2017; 46: 505-529
• 26 Mei Y, Wang Y, Chen H. et al. Recent progress in CRISPR/Cas9 technology. J Genet Genomics 2016; 43: 63-75
• 27 Ran FA, Hsu PD, Wright J. et al. Genome engineering using the CRISPR-Cas9 system. Nat Protoc 2013; 8: 2281-2308
• 28 Fagerlund RD, Wilkinson ME, Klykov O. et al. Spacer capture and integration by a type I-F Cas1-Cas2–3 CRISPR adaptation complex. Proc Natl Acad Sci U S A 2017; 114: E5122-E5128
• 29 Pourcel C, Salvignol G, Vergnaud G. CRISPR elements in yersinia pestis acquire new repeats by preferential uptake of bacteriophage DNA, and provide additional tools for evolutionary studies. Microbiology 2005; 151: 653-663
• 30 Nuñez JK, Lee AS, Engelman A. et al. Integrase-mediated spacer acquisition during CRISPR-Cas adaptive immunity. Nature 2015; 519: 193-198
• 31 Brouns SJ, Jore MM, Lundgren M. et al. Small CRISPR RNAs guide antiviral defense in prokaryotes. Science 2008; 321: 960-964
• 32 Shen S, Loh TJ, Shen H. et al. CRISPR as a strong gene editing tool. BMB Rep 2017; 50: 20-24
• 33 Terao M, Tamano M, Hara S. et al. Utilization of the CRISPR/Cas9 system for the efficient production of mutant mice using crRNA/tracrRNA with Cas9 nickase and fokI-dCas9. Exp Anim 2016; 65: 275-283
• 34 Westra ER, van Erp PB, Künne T. et al. CRISPR immunity relies on the consecutive binding and degradation of negatively supercoiled invader DNA by Cascade and Cas3. Molecular Cell 2012; 46: 595-605
• 35 Huai C, Li G, Yao R. et al. Structural insights into DNA cleavage activation of CRISPR-Cas9 system. Nat Commun 2017; 8: 1375
• 36 Doudna JA, Charpentier E. Genome editing. The new frontier of genome engineering with CRISPR-Cas9. Science 2014; 346: 1258096
• 37 Williams BO, Warman ML. CRISPR/Cas9 Technologies. J Bone Miner Res 2017; 32: 883-888
• 38 Choi JG, Dang Y, Abraham S. et al. Lentivirus pre-packed with Cas9 protein for safer gene editing. Gene Ther 2016; 23: 627-633
• 39 Puschnik AS, Majzoub K, Ooi YS. et al. A CRISPR toolbox to study virus-host interactions. Nat Rev Microbiol 2017; 15: 351-364
• 40 Peng YQ, Tang LS, Yoshida S. et al. Applications of CRISPR/Cas9 in retinal degenerative diseases. Int J Ophthalmol 2017; 10: 646-651
• 41 Yang D, Song J, Zhang J. et al. Identification and characterization of rabbit rosa26 for gene knock-in and stable reporter gene expression. Sci Rep 2016; 6: 25161
• 42 Wang B, Li K, Wang A. et al. Highly efficient CRISPR/HDR-mediated knock-in for mouse embryonic stem cells and zygotes. Biotechniques 2015; 59: 201-202 204, 206–208
• 43 Chen YJ, Cheng YY, Wang W. et al. Rapid, modular, and cost-effective generation of donor DNA constructs for CRISPR-based gene knock-in. Biol Methods Protoc 2020; 5: bpaa006
• 44 Sakuma T, Yamamoto T. Magic wands of CRISPR-lots of choices for gene knock-in. Cell Biol Toxicol 2017; 33: 501-505
• 45 Suzuki K, Tsunekawa Y, Hernandez-Benitez R. et al. In vivo genome editing via CRISPR/Cas9 mediated homology-independent targeted integration. Nature 2016; 540: 144-149
• 46 Long C, McAnally JR, Shelton JM. et al. Prevention of muscular dystrophy in mice by CRISPR/Cas9-mediated editing of germline DNA. Science 2014; 345: 1184-1188
• 47 Vyas VK, Barrasa MI, Fink GR. A Candida albicans CRISPR system permits genetic engineering of essential genes and gene families. Sci Adv 2015; 1: e1500248
• 48 Komor AC, Kim YB, Packer MS. et al. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature 2016; 533: 420-424
• 49 Qi LS, Larson MH, Gilbert LA. et al. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 2013; 152: 1173-1183
• 50 Brocken DJW, Tark-Dame M, Dame RT. dCas9: A versatile tool for epigenome editing. Curr Issues Mol Biol 2018; 26: 15-32
• 51 Ye H, Rong Z, Lin Y. Live cell imaging of genomic loci using dCas9-SunTag system and a bright fluorescent protein. Protein Cell 2017; 8: 853-855
• 52 Ma H, Tu LC, Chung YC. et al. Cell cycle- and genomic distance-dependent dynamics of a discrete chromosomal region. J Cell Biol 2019; 218: 1467-1477
• 53 Maass PG, Barutcu AR, Weiner CL. et al. Inter-chromosomal Contact Properties in Live-Cell Imaging and in Hi-C. Mol Cell 2018; 69: 1039-1045.e3
• 54 Fu Y, Sander JD, Reyon D. et al. Improving CRISPR-Cas nuclease specificity using truncated guide RNAs. Nat Biotechnol 2014; 32: 279-284
• 55 Zhang XH, Tee LY, Wang XG. et al. Off-target effects in CRISPR/Cas9-mediated genome engineering. Mol Ther Nucleic Acids 2015; 4: e264
• 56 Li D, Wang H, Song H. et al. The microRNAs mir-200b-3p and miR-429–5p target the LIMK1/CFL1 pathway to inhibit growth and motility of breast cancer cells. Oncotarget 2017; 8: 85276-85289
• 57 Sun G, Cao Y, Wang P. et al. miR-200b-3p in plasma is a potential diagnostic biomarker in oral squamous cell carcinoma. Biomarkers 2018; 23: 137-141
• 58 Wu J, Tao Y, Shang A. et al. Effect of the interaction between mir-200b-3p and dnmt3a on cartilage cells of osteoarthritis patients. J Cell Mol Med 2017; 21: 2308-2316
• 59 Chen B, Zou W, Xu H. et al. Efficient labeling and imaging of protein-coding genes in living cells using CRISPR-Tag. Nat Commun 2018; 9: 5065
• 60 Zengini E, Hatzikotoulas K, Tachmazidou I. et al. Genome-wide analyses using UK biobank data provide insights into the genetic architecture of osteoarthritis. Nat Genet 2018; 50: 549-558
• 61 Klein JC, Keith A, Rice SJ. et al. Functional testing of thousands of osteoarthritis-associated variants for regulatory activity. Nat Commun 2019; 10: 2434
• 62 Castaño-Betancourt MC, Evans DS, Ramos YF. et al. Novel genetic variants for cartilage thickness and hip osteoarthritis. PLoS Genet 2016; 12: e1006260
• 63 Rice SJ, Aubourg G, Sorial AK. et al. Identification of a novel, methylation-dependent, RUNX2 regulatory region associated with osteoarthritis risk. Hum Mol Genet 2018; 27: 3464-3474
• 64 Austin CP, Battey JF, Bradley A. et al. The knockout mouse project. Nat Genet 2004; 36: 921-924
• 65 Little CB, Hunter DJ. Post-traumatic osteoarthritis: From mouse models to clinical trials. Nat Rev Rheumatol 2013; 9: 485-497
• 66 Chen M, Lichtler AC, Sheu TJ. et al. Generation of a transgenic mouse model with chondrocyte-specific and tamoxifen-inducible expression of Cre recombinase. Genesis 2007; 45: 44-50
• 67 Luo Y, Sinkeviciute D, He Y. et al. The minor collagens in articular cartilage. Protein Cell 2017; 8: 560-572
• 68 Shwartz Y, Viukov S, Krief S. et al. Joint Development Involves a Continuous Influx of Gdf5-Positive Cells. Cell Rep 2016; 15: 2577-2587
• 69 Alayoubi AM, Khawaji ZY, Mohammed MA. et al. CRISPR-Cas9 system: a novel and promising era of genotherapy for beta-hemoglobinopathies, hematological malignancy, and hemophilia. Ann Hematol 2024; 103: 1805-1817
• 70 Khouzam JPS, Tivakaran VS. CRISPR-Cas9 Applications in Cardiovascular Disease. Curr Probl Cardiol 2021; 46: 100652
• 71 Powell SK, Gregory J, Akbarian S. et al. Application of CRISPR/Cas9 to the study of brain development and neuropsychiatric disease. Mol Cellular Neurosci 2017; 82: 157-166
• 72 Pierce EA, Aleman TS, Jayasundera KT. et al. Gene Editing for CEP290-Associated Retinal Degeneration. N Engl J Med 2024; 390: 1972-1984
• 73 Zhao L, Huang J, Fan Y. et al. Exploration of CRISPR/Cas9-based gene editing as therapy for osteoarthritis. Ann Rheum Dis 2019; 78: 676-682
• 74 Seidl CI, Fulga TA, Murphy CL. CRISPR-Cas9 targeting of MMP13 in human chondrocytes leads to significantly reduced levels of the metalloproteinase and enhanced type II collagen accumulation. Osteoarthritis Cartilage 2019; 27: 140-147
• 75 Varela-Eirín M, Varela-Vázquez A, Guitián-Caamaño A. et al. Targeting of chondrocyte plasticity via connexin43 modulation attenuates cellular senescence and fosters a pro-regenerative environment in osteoarthritis. Cell Death Dis 2018; 9: 1166
• 76 Rai MF, Sandell LJ. Emerging concepts in gene therapy for osteoarthritis. J Am Acad Orthop Surg 2015; 23: e56-e57
• 77 Evans CH, Ghivizzani SC, Robbins PD. Gene delivery to joints by intra-articular injection. Hum Gene Ther 2018; 29: 2-14
• 78 Lu Y, Godbout K, Lamothe G. et al. CRISPR-Cas9 delivery strategies with engineered extracellular vesicles. Mol Ther Nucleic Acids 2023; 34: 102040
• 79 Chen M, Lu Y, Liu Y. et al. Injectable Microgels with Hybrid Exosomes of Chondrocyte-Targeted FGF18 Gene-Editing and Self-Renewable Lubrication for Osteoarthritis Therapy. Adv Mater 2024; 36: e2312559
• 80 Mangeot PE, Dollet S, Girard M. et al. Protein transfer into human cells by VSV-G-induced nanovesicles. Mol Ther 2011; 19: 1656-1666
• 81 Campbell LA, Coke LM, Richie CT. et al. Gesicle-mediated delivery of CRISPR/Cas9 ribonucleoprotein complex for inactivating the HIV provirus. Mol Ther 2019; 27: 151-163
• 82 Jing Y, Jiang X, Ji Q. et al. Genome-wide CRISPR activation screening in senescent cells reveals SOX5 as a driver and therapeutic target of rejuvenation. Cell Stem Cell 2023; 30: 1452-1471
• 83 Chen M, Lu Y, Liu Y. et al. Injectable Microgels with Hybrid Exosomes of Chondrocyte-Targeted FGF18 Gene-Editing and Self-Renewable Lubrication for Osteoarthritis Therapy. Adv Mater 2024; 36: e2312559
• 84 Zhao L, Lai Y, Jiao H. et al. CRISPR-mediated Sox9 activation and RelA inhibition enhance cell therapy for osteoarthritis. Mol Ther 2024; 32: 2549-2562
• 85 Wang KD, Ding X, Jiang N. et al. Digoxin targets low density lipoprotein receptor-related protein 4 and protects against osteoarthritis. Ann Rheum Dis 2022; 81: 544-555
• 86 Kuang B, Geng N, Yi M. et al. Panaxatriol exerts anti-senescence effects and alleviates osteoarthritis and cartilage repair fibrosis by targeting UFL1 . J Adv Res 2024;
• 87 Liang Y, Xu X, Xu L. et al. Chondrocyte-specific genomic editing enabled by hybrid exosomes for osteoarthritis treatment. Theranostics 2022; 12: 4866-4878
• 88 Wang W, Zhu Y, Sun Z. et al. Positive feedback regulation between USP15 and ERK2 inhibits osteoarthritis progression through TGF-β/SMAD2 signaling. Arthritis Res Ther 2021; 23: 84
• 89 Murakami T, Ruengsinpinya L, Takahata Y. et al. HOXA10 promotes Gdf5 expression in articular chondrocytes. Sci Rep 2023; 13: 22778
• 90 Fu L, Hu Y, Song M. et al. Up-regulation of FOXD1 by YAP alleviates senescence and osteoarthritis. PLoS Biol 2019; 17: e3000201
• 91 Ren X, Hu B, Song M. et al. Maintenance of Nucleolar Homeostasis by CBX4 Alleviates Senescence and Osteoarthritis. Cell Rep 2019; 26: 3643-3656.e7
• 92 Bloks NG, Harissa Z, Mazzini G. et al. A Damaging COL6A3 Variant Alters the MIR31HG-Regulated Response of Chondrocytes in Neocartilage Organoids to Hyperphysiologic Mechanical Loading. Adv Sci (Weinh) 2024; 11: e2400720
• 93 Yammine KM, Mirda Abularach S, Sampurno L. et al. Using CRISPR/Cas9 to generate a heterozygous COL2A1 p.R719C iPSC line (MCRIi019-A-6) model of human precocious osteoarthritis. Stem Cell Res 2023; 67: 103020
• 94 Bonato A, Fisch P, Ponta S. et al. Engineering Inflammation-Resistant Cartilage: Bridging Gene Therapy and Tissue Engineering. Adv Healthc Mater 2023; 12: e2202271
• 95 Varela-Eirín M, Varela-Vázquez A, Guitián-Caamaño A. et al. Targeting of chondrocyte plasticity via connexin43 modulation attenuates cellular senescence and fosters a pro-regenerative environment in osteoarthritis. Cell Death Dis 2018; 9: 1166
• 96 Sorial AK, Hofer IMJ, Tselepi M. et al. Multi-tissue epigenetic analysis of the osteoarthritis susceptibility locus mapping to the plectin gene PLEC . Osteoarthritis Cartilage 2020; 28: 1448-1458
• 97 Chaudhry N, Muhammad H, Seidl C. et al. Highly efficient CRISPR-Cas9-mediated editing identifies novel mechanosensitive microRNA-140 targets in primary human articular chondrocytes. Osteoarthritis Cartilage 2022; 30: 596-604
• 98 Zhang J, Liao JQ, Wen LR. et al. Rps6ka2 enhances iMSC chondrogenic differentiation to attenuate knee osteoarthritis through articular cartilage regeneration in mice. Biochem Biophys Res Commun 2023; 663: 61-70
• 99 Raman R, Bahri MA, Degueldre C. et al. A Zebrafish Mutant in the Extracellular Matrix Protein Gene GRASLND as a Model for Spinal Osteoarthritis. Animals (Basel) 2023; 14: 74
• 100 Kung LHW, Sampurno L, Little CB. et al. Generation of a miR-26b stem-loop knockout human iPSC line, MCRIi019-A-1, using CRISPR/Cas9 editing. Stem Cell Res 2020; 50: 102118
• 101 Dreier R, Ising T, Ramroth M. et al. Estradiol Inhibits ER Stress-Induced Apoptosis in Chondrocytes and Contributes to a Reduced Osteoarthritic Cartilage Degeneration in Female Mice. Front Cell Dev Biol 2022; 10: 913118
• 102 Brunger JM, Zutshi A, Willard VP. et al. Genome Engineering of Stem Cells for Autonomously Regulated, Closed-Loop Delivery of Biologic Drugs. Stem Cell Reports 2017; 8: 1202-1213
• 103 D’Costa S, Rich MJ, Diekman BO. Engineered Cartilage from Human Chondrocytes with Homozygous Knockout of Cell Cycle Inhibitor p21. Tissue Eng Part A 2020; 26: 441-449
• 104 Okutani Y, Abe K, Yamashita A. et al. Generation of Monkey Induced Pluripotent Stem Cell-Derived Cartilage Lacking Major Histocompatibility Complex Class I Molecules on the Cell Surface. Tissue Eng Part A 2022; 28: 94-106
• 105 Huynh NP, Gloss CC, Lorentz J. et al. Long non-coding RNA GRASLND enhances chondrogenesis via suppression of the interferon type II signaling pathway. Elife 2020; 9: e49558