Fig. 1

Overview of the experimental workflow outlined in this study. A Transfection optimization in primary polydactyly chondrocytes was performed by delivering a green fluorescent protein (GFP)-labelled Cas9-RNP targeting the housekeeping hypoxanthine guanine phosphoribosyltransferase (HPRT) gene, comparing electroporation and lipid nanoparticle delivery. After 24 h, microscopy imaging was used to infer transfection-associated cytotoxicity and efficiency, by counting live, dead and GFP⁺ cells, respectively. At 48 h post-delivery, DNA was extracted and the HPRT locus was amplified by polymerase chain reaction (PCR). Upon denaturation and re-annealing, mismatched heteroduplexes arising from base-pairing of WT and edited alleles were cut using the T7 endonuclease 1 (T7E1) enzyme. The generated fragments were run on a 1.2% agarose gel and editing efficiency was calculated. B Reagents optimization was carried out by testing three different Cas9 enzymes and two gRNA formulations, respectively. Editing efficiency at the HPRT locus in polydactyly chondrocytes was validated with Sanger sequencing. C Using our optimized parameters and reagents, we generated bulk-edited chondrocyte populations harboring a RELA KO. RELA is an essential component of the NF-κB complex, which regulates the activation of several pro-inflammatory cellular pathways (e.g., interleukin-1 beta (IL-1β), MMP13 and tumor necrosis factor alpha (TNFα)). Besides calculating editing efficiency, we further characterized KO polydactyly chondrocytes with quantitative reverse transcription PCR (RT-qPCR) and Western Blot. Applicability and reproducibility of our KO gene editing technique was further tested in OA-patient-derived chondrocytes, FE002 primary chondroprogenitors, a human chondrocyte cell line and bovine chondrocytes, with RELA KO efficiency calculated using Sanger sequencing and phenotype validated via qPCR. D WT and KO pellets for all cell types were cultured for 3 weeks in chondrogenic media, with and without the addition of 10 ng/ml of IL-1β throughout the last week of culture. Extracellular matrix (ECM) deposition was assessed using histological staining for detecting glycosaminoglycans (GAGs) and immunostaining to verify the deposition of collagen type I and type II fibers