Osteoarthritis (OA) is a chronic degenerative joint disease. Currently, OA is incurable. Abnormal activation of canonical Wnt/β-catenin or Indian hedgehog (Ihh) signaling could lead to OA development and progression. This study aimed to determine if targeting β-catenin and Ihh signaling could yield an effective therapeutic intervention for OA disease. CRISPR/CasRx is a new RNA interference tool that can precisely and efficiently cleave single-strand RNAs. In this study, we screened CRISPR-derived RNA (crRNA) targeting Ctnnb1 and Smo in vitro and selected two optimal crRNAs for each gene. CasRx-mediated Ctnnb1 and Smo knockdown showed high efficiency and specificity with no obvious off-target effects in vitro. We then performed intra-articular injection of selected crRNAs driven by the adeno-associated virus into an OA mouse model. Micro-CT, histological, and histomorphometric analyses were conducted to evaluate the efficacy of CasRx approach on OA treatment. We found that the knockdown of Ctnnb1 and Smo decelerated pathological damage in the keen joint of the experimental OA mouse model. Our findings suggest that CasRx-mediated Ctnnb1 and Smo knockdown could be a potential strategy for OA treatment.