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.

中文翻译：

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高效 CRISPR-Cas9 介导的编辑可识别原代人关节软骨细胞中的新型机械敏感 microRNA-140 靶标

客观的

MicroRNA 140 ( miR-140)是一种与骨关节炎 (OA) 相关的软骨细胞特异性内源性基因调节剂。由于机械损伤是 OA 的主要病因，我们在原代人软骨细胞中使用新型 CRISPR-Cas9 方法研究了miR-140依赖性机械敏感基因调控。

方法

原代（1/2 代）人 OA 软骨细胞从关节成形术样本（6 个供体）中分离出来，并使用靶向miR-140 的单向导 RNA (sgRNA) 结合 Cas9 核酸内切酶，用核糖核蛋白复合物或质粒转染。测试了 sgRNA 和单/双转染的组合。通过 T7 核酸内切酶 1 (T7E1) 测定法测量基因编辑。miRNA 水平通过 qPCR 在软骨细胞和急性损伤后的野生型鼠股骨头软骨中证实。检查预测的近距离匹配脱靶。机械敏感的miR-140使用 TaqMan 微流体卡在靶向和供体匹配的对照软骨细胞中评估 42 个损伤相关基因的靶标验证。在来自miR-140 ^-/-小鼠的肋软骨细胞的 RNAseq 数据中检查了已识别的目标。

结果

当两个 sgRNA 与双 RNP 介导的 CRISPR-Cas9 转染相结合时，获得了miR-140的高效基因编辑(90–98%)。股骨软骨损伤后miR-140水平迅速下降。在确定的前八个miR-140基因靶点中 ( P  < 0.01)，我们验证了三个先前确定的靶点（septin 2、骨形态发生蛋白 2 和成纤维细胞生长因子 2）。新靶点包括Agrin（一种新识别的促再生软骨剂），以及与视黄酸信号和初级纤毛相关的蛋白质。

结论

我们描述了一种高效的 CRISPR-Cas9 介导的基因编辑策略，用于原代人软骨细胞的基因编辑，并确定了几个与疾病相关的新型机械敏感miR-140靶标。