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. 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.