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

Lookup NU author(s): Professor David YoungORCiD, Yao Hao

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This work is licensed under a Creative Commons Attribution 4.0 International License (CC BY 4.0).


Abstract

© 2022 The Authors. Objective: MicroRNA 140 (miR-140) is a chondrocyte-specific endogenous gene regulator implicated in osteoarthritis (OA). As mechanical injury is a primary aetiological factor in OA, we investigated miR-140-dependent mechanosensitive gene regulation using a novel CRISPR-Cas9 methodology in primary human chondrocytes. Method: Primary (passage 1/2) human OA chondrocytes were isolated from arthroplasty samples (six donors) and transfected with ribonuclear protein complexes or plasmids using single guide RNAs (sgRNAs) targeting miR-140, in combination with Cas9 endonuclease. Combinations of sgRNAs and single/double transfections were tested. Gene editing was measured by T7 endonuclease 1 (T7E1) assay. miRNA levels were confirmed by qPCR in chondrocytes and in wild type murine femoral head cartilage after acute injury. Predicted close match off-targets were examined. Mechanosensitive miR-140 target validation was assessed in 42 injury-associated genes using TaqMan Microfluidic cards in targeted and donor-matched control chondrocytes. Identified targets were examined in RNAseq data from costal chondrocytes from miR-140−/− mice. Results: High efficiency gene editing of miR-140 (90–98%) was obtained when two sgRNAs were combined with double RNP-mediated CRISPR-Cas9 transfection. miR-140 levels fell rapidly after femoral cartilage injury. Of the top eight miR-140 gene targets identified (P < 0.01), we validated three previously identified ones (septin 2, bone morphogenetic protein 2 and fibroblast growth factor 2). Novel targets included Agrin, a newly recognised pro-regenerative cartilage agent, and proteins associated with retinoic acid signalling and the primary cilium. Conclusion: We describe a highly efficient CRISPR-Cas9-mediated strategy for gene editing in primary human chondrocytes and identify several novel mechanosensitive miR-140 targets of disease relevance.


Publication metadata

Author(s): Chaudhry N, Muhammad H, Seidl C, Downes D, Young DA, Hao Y, Zhu L, Vincent TL

Publication type: Article

Publication status: Published

Journal: Osteoarthritis and Cartilage

Year: 2022

Pages: Epub ahead of print

Online publication date: 21/01/2022

Acceptance date: 14/01/2022

Date deposited: 07/03/2022

ISSN (print): 1063-4584

ISSN (electronic): 1522-9653

Publisher: Elsevier Ltd.

URL: https://doi.org/10.1016/j.joca.2022.01.005

DOI: 10.1016/j.joca.2022.01.005

PubMed id: 35074547


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Funding

Funder referenceFunder name
20205
21621
R476/0516Dunhill Medical Trust

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