Dataset Information
Highly efficient CRISPR-Cas9-mediated editing identifies novel mechanosensitive microRNA-140 targets in primary human articular chondrocytes.
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ABSTRACT: 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.
SUBMITTER: Chaudhry N
PROVIDER: S-EPMC8987936 | biostudies-literature | 2022 Apr
REPOSITORIES: biostudies-literature
Publications
Highly efficient CRISPR-Cas9-mediated editing identifies novel mechanosensitive microRNA-140 targets in primary human articular chondrocytes.
Osteoarthritis and cartilage 20220121 4
<h4>Objective</h4>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.<h4>Method</h4>Primary (passage 1/2) human OA chondrocytes were isolated from arthroplasty samples (six donors) and transfected with ribonuclear protein complexes or plasmids u ...[more]