Hlynur Magnússon and Sara Sigurbjörnsdóttir
Introduction
Osteoarthritis (OA), a prevalent joint disease influenced by genetic and environmental factors, is characterised by the progressive deterioration of cartilage in joints like the hip, knee, or hands. The SMO protein plays a pivotal role in activating the hedgehog signalling pathway, a process linked to the differentiation of chondrocytes. Our study focuses on a missense mutation in the human Smoothened (SMO) gene, specifically the substitution of arginine (R) at position 173 with cysteine (A). Numerous genome-wide association studies (GWAS) indicate that this mutation significantly heightens the risk of OA in carriers.
Method
Our study employed an optimised CRISPR/Cas9 technique, a cutting-edge tool in genetic research, to establish a genetically modified cell line endogenously expressing the Smo variant. A single-stranded template containing the variant was designed and introduced to the genome with homology-directed repair (HDR) induced by Cas9. Nocodazole, a microtubule inhibitor, was used to arrest the cells in the appropriate phase where HDR is activated. Expression of Smo and other OA factors were analysed using quantitative polymerase chain reaction.
Results
Our optimised protocol generating a chondrocyte cell line that endogenously expresses the mutated Smo variant was a success. Assays indicate increased expression of Smo in the R173C cell line compared to SMO-deficient cells.
Conclusion
Creating the R173C cell line allows us to understand further SMO and the variant’s role in OA progression, which could lead to further treatment possibilities.
