Differential expression of RhoA gene in SMART-Cas9 knockout macrophages
We have identified that Ras homolog gene family member A (RhoA) is a pivotal target in inducing osteoclastogenesis in macrophages. Subsequently, we developed a strategy termed Specific Macrophage RhoA Targeting (SMART). In this strategy, phosphatidylserine-enriched macrophage membranes are engineered to deliver CRISPR-Cas9 plasmids containing a macrophage-specific promoter (SMART-Cas9), thereby enabling targeted editing of the RhoA gene in RAW264.7 macrophages. High-throughput transcriptome sequencing was then performed. Transcriptome analysis revealed that ablation of the macrophage RhoA gene significantly inhibited key signaling pathways associated with bone destruction. Overall design: To investigate the effect of macrophage RhoA deficiency on bone destruction in rheumatoid arthritis (RA), we employed a targeted macrophage-specific RhoA knockout strategy using SMART-Cas9. One untreated macrophage cell line was used as the control group. Subsequently, total RNA was extracted from the cells, and high-throughput transcriptome sequencing was performed. Gene expression profiling was then conducted using the data obtained from RNA-seq. Each sample was replicated three times to ensure robustness and reproducibility of the results.