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. 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). 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. 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.

中文翻译：

---

简化的单步非病毒 CRISPR-Cas9 敲除策略提高了原代人类软骨细胞群的基因编辑效率

基于 CRISPR-Cas9 的基因组工程代表了软骨组织工程和了解驱动软骨疾病的分子途径的强大治疗工具。然而，原代软骨细胞在单层（2D）细胞培养过程中难以转染和快速去分化，使得单细胞衍生的编辑克隆群体的长时间扩增不可行。因此，长期以来，针对软骨和风湿性疾病的功能遗传学研究一直在细胞模型中进行，这些模型很难概括人类软骨组织的天然分子特性（例如细胞系、诱导多能干细胞）。在这里，我们着手开发一种非病毒 CRISPR-Cas9 批量基因编辑方法，适用于不同软骨来源的软骨细胞群。我们筛选了用于原代多指软骨细胞中核糖核蛋白 (RNP) 递送的电穿孔和脂质纳米粒子，并优化了 RNP 试剂组装。我们在多指软骨细胞中敲除了 RELA（也称为 p65），它是核因子 kappa B (NF-κB) 的一个亚基，并通过 RT-qPCR 和 Western Blot 进一步表征了敲除 (KO) 细胞。我们在不同软骨来源的软骨细胞中测试了 RELA KO，并通过 RT-qPCR 表征了它们的表型。我们检测了存在和不存在白介素-1β (IL-1β) 的情况下野生型 (WT) 和 KO 细胞沉淀的软骨形成潜力。我们将电穿孔确立为软骨细胞的最佳转染技术，可增强转染和编辑效率，同时保持高细胞活力。我们以约 90% 的前所未有的效率敲除 RELA，证实与未经编辑的细胞相比，IL-1β 刺激后炎症通路的激活较低。我们的方案可以轻松转移到从骨关节炎 (OA) 患者、人 FE002 软骨祖细胞、牛软骨细胞和人软骨细胞细胞系中收获的原代人软骨细胞，使用相同的方案实现可比较的平均 RELA KO 编辑水平。来自原代人软骨细胞的所有 KO 颗粒都保留了与 WT 细胞相当的软骨形成能力，并且在炎症条件下还表现出增强的基质保留能力。我们展示了我们的批量基因编辑方法的适用性，以开发有效的自体和同种异体现成基因治疗策略，并使人类软骨细胞的功能遗传学研究能够阐明软骨疾病的分子机制。