Highly efficient CRISPR-Cas9-mediated editing identifies novel mechanosensitive microRNA-140 targets in primary human articular chondrocytes. Issue 4 (April 2022)

• Record Type: Journal Article
• Authors: Chaudhry, N.; Muhammad, H.; Seidl, C.; Downes, D.; Young, D.A.; Hao, Y.; Zhu, L.; Vincent, T.L.
• Is Part Of: Osteoarthritis and cartilage. Volume 30:Issue 4(2022)
• Page Start: 596
• Page End: 604
• Publication Date: 2022-04
• Subjects: Chondrocyte -- Human -- Osteoarthritis -- CRISPR-Cas9 -- miR-140 -- Injury; Osteoarthritis -- Periodicals; Cartilage -- Periodicals; Osteoarthritis -- Periodicals; Cartilage -- Periodicals; Arthrose -- Périodiques; Articulations -- Maladies -- Périodiques; 616.7223005
• DOI: 10.1016/j.joca.2022.01.005
• Languages: English
• ISSNs: 1063-4584

Abstract

Summary: 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.