Highly efficient CRISPR-Cas9-mediated editing identifies novel mechanosensitive microRNA-140 targets in primary human articular chondrocytes

Chaudhry, Noman; Muhammad, Hayat; Seidl, Christine; Downes, Damien J.; Young, David A.; Yao, Hao; Zhu, Longji; Vincent, Tonia L.

doi:10.1016/j.joca.2022.01.005

Cited by 9 publications

(6 citation statements)

References 53 publications

(56 reference statements)

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“…Previous gene editing efforts in primary chondrocytes reported a lower gene editing efficiency, reaching 74% efficiency with MMP13 to reduce ECM degradation, [47] or 60% for cell cycle inhibitor p21 to increase ECM deposition, [49] of which only 16% of colonies showed a homozygous deletion. Similar efficiency has only been demonstrated in a recent work targeting miR-140, where 90-98% of editing was reached; [48] yet that required two sgRNAs and double transfection, conditions that might enhance post-transfection cell toxicity and off-target probability, even if not observed by T7E1 assay. In our editing, we observed a 60% deletion of the first predicted off-target.…”

Section: Discussionsupporting

confidence: 80%

“…The system yielded a high efficiency in production of genome-edited cells across several donors, which was superior to previous attempts. [47][48][49] Next, we characterized the gene expression and matrix deposition of the TAK1-KO chondrocytes exposed to inflammatory stimuli. Then the ability of TAK1-KO chondrocytes to produce good quality cartilage in a hydrogel when exposed to an inflamed environment was assessed.…”

Section: Introductionmentioning

confidence: 99%

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Engineering Inflammation‐Resistant Cartilage: Bridging Gene Therapy and Tissue Engineering

Bonato

Fisch

Ponta

et al. 2023

Adv Healthcare Materials

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Articular cartilage defects caused by traumatic injury rarely heal spontaneously and predispose into post-traumatic osteoarthritis. In the current autologous cell-based treatments the regenerative process is often hampered by the poor regenerative capacity of adult cells and the inflammatory state of the injured joint. The lack of ideal treatment options for cartilage injuries motivated the authors to tissue engineer a cartilage tissue which would be more resistant to inflammation. A clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 knockout of TGF-𝜷-activated kinase 1 (TAK1) gene in polydactyly chondrocytes provides multivalent protection against the signals that activate the pro-inflammatory and catabolic NF-𝜿B pathway. The TAK1-KO chondrocytes encapsulate into a hyaluronan hydrogel deposit copious cartilage extracellular matrix proteins and facilitate integration onto native cartilage, even under proinflammatory conditions. Furthermore, when implanted in vivo, compared to WT fewer pro-inflammatory M1 macrophages invade the cartilage, likely due to the lower levels of cytokines secreted by the TAK1-KO polydactyly chondrocytes. The engineered cartilage thus represents a new paradigm-shift for the creation of more potent and functional tissues for use in regenerative medicine.

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Section: Discussionsupporting

confidence: 80%

Section: Introductionmentioning

confidence: 99%

Engineering Inflammation‐Resistant Cartilage: Bridging Gene Therapy and Tissue Engineering

Bonato

Fisch

Ponta

et al. 2023

Adv Healthcare Materials

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“…Here, we developed an RNP gene editing strategy to achieve strikingly high editing efficiency in bulk, generating a chondrocyte cell pool ready for downstream application, without the need for further enrichment. Our KO efficiency is considerably greater than in reported RNP analogous approaches [ 27 , 28 ], and comparable to a recent work published by our group [ 29 ]. RNP delivery ensures transient editing, thereby reducing the risk of off-target effects and immunogenicity.…”

Section: Discussionsupporting

confidence: 90%

“…RNP-based bulk gene editing in primary chondrocytes has been performed to KO matrix metallopeptidase 13 ( MMP13 ), with editing efficiency ranging from 63 to 74% [ 27 ]. Higher editing percentages (~ 90–98%) were achieved only upon delivery of two sgRNAs and a double transfection to target microRNA 140 [ 28 ].…”

Section: Introductionmentioning

confidence: 99%

Streamlined, single-step non-viral CRISPR-Cas9 knockout strategy enhances gene editing efficiency in primary human chondrocyte populations

Ponta,

Bonato,

Neidenbach

et al. 2024

Arthritis Res Ther

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Background CRISPR-Cas9-based genome engineering represents a powerful therapeutic tool for cartilage tissue engineering and for understanding molecular pathways driving cartilage diseases. However, primary chondrocytes are difficult to transfect and rapidly dedifferentiate during monolayer (2D) cell culture, making the lengthy expansion of a single-cell-derived edited clonal population not feasible. For this reason, functional genetics studies focused on cartilage and rheumatic diseases have long been carried out in cellular models that poorly recapitulate the native molecular properties of human cartilaginous tissue (e.g., cell lines, induced pluripotent stem cells). Here, we set out to develop a non-viral CRISPR-Cas9, bulk-gene editing method suitable for chondrocyte populations from different cartilaginous sources. Methods We screened electroporation and lipid nanoparticles for ribonucleoprotein (RNP) delivery in primary polydactyly chondrocytes, and optimized RNP reagents assembly. We knocked out RELA (also known as p65), a subunit of the nuclear factor kappa B (NF-κB), in polydactyly chondrocytes and further characterized knockout (KO) cells with RT-qPCR and Western Blot. We tested RELA KO in chondrocytes from diverse cartilaginous sources and characterized their phenotype with RT-qPCR. We examined the chondrogenic potential of wild-type (WT) and KO cell pellets in presence and absence of interleukin-1β (IL-1β). Results We established electroporation as the optimal transfection technique for chondrocytes enhancing transfection and editing efficiency, while preserving high cell viability. We knocked out RELA with an unprecedented efficiency of ~90%, confirming lower inflammatory pathways activation upon IL-1β stimulation compared to unedited cells. Our protocol could be easily transferred to primary human chondrocytes harvested from osteoarthritis (OA) patients, human FE002 chondroprogenitor cells, bovine chondrocytes, and a human chondrocyte cell line, achieving comparable mean RELA KO editing levels using the same protocol. All KO pellets from primary human chondrocytes retained chondrogenic ability equivalent to WT cells, and additionally displayed enhanced matrix retention under inflamed conditions. Conclusions We showcased the applicability of our bulk gene editing method to develop effective autologous and allogeneic off-the-shelf gene therapies strategies and to enable functional genetics studies in human chondrocytes to unravel molecular mechanisms of cartilage diseases.

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“…In humans, gene editing has been performed using the CRISPR/Cas9 method, in order to silence miR-140-5p/-3p expression in primary OA chondrocytes, without WWP2 expression modulation. This recent development has only been used at this stage to identify miR-140-5p/-3p targets, but opens up great possibilities as a tool to further evaluate the role of miRNAs in AO and to assess potential new therapies [ 109 ].…”

Section: Role Of Mir-140-5p/-3p In Osteoarthritismentioning

confidence: 99%

miR-140-5p and miR-140-3p: Key Actors in Aging-Related Diseases?

Toury

Frankel

Airault

et al. 2022

IJMS

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microRNAs (miRNAs) are small single strand non-coding RNAs and powerful gene expression regulators. They mainly bind to the 3′UTR sequence of targeted mRNA, leading to their degradation or translation inhibition. miR-140 gene encodes the pre-miR-140 that generates the two mature miRNAs miR-140-5p and miR-140-3p. miR-140-5p/-3p have been associated with the development and progression of cancers, but also non-neoplastic diseases. In aging-related diseases, miR-140-5p and miR-140-3p expressions are modulated. The seric levels of these two miRNAs are used as circulating biomarkers and may represent predictive tools. They are also considered key actors in the pathophysiology of aging-related diseases. miR-140-5p/-3p repress targets regulating cell proliferation, apoptosis, senescence, and inflammation. This work focuses on the roles of miR-140-3p and miR-140-5p in aging-related diseases, details their regulation (i.e., by long non-coding RNA), and reviews the molecular targets of theses miRNAs involved in aging pathophysiology.

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Highly efficient CRISPR-Cas9-mediated editing identifies novel mechanosensitive microRNA-140 targets in primary human articular chondrocytes

Cited by 9 publications

References 53 publications

Engineering Inflammation‐Resistant Cartilage: Bridging Gene Therapy and Tissue Engineering

Engineering Inflammation‐Resistant Cartilage: Bridging Gene Therapy and Tissue Engineering

Streamlined, single-step non-viral CRISPR-Cas9 knockout strategy enhances gene editing efficiency in primary human chondrocyte populations

miR-140-5p and miR-140-3p: Key Actors in Aging-Related Diseases?

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