## 3 Results
### Gene Therapy and RNA Interference (RNAi)
#### 3.3.2 CRISPR-Cas9  
In recent years, CRISPR-Cas9 technology has transformed the field of genetic engineering, enabling precise and efficient modification of DNA sequences across a wide array of organisms. Discovered as part of the adaptive immune system in bacteria and archaea, CRISPR-Cas9 functions by introducing site-specific breaks in DNA, allowing for targeted gene knockout, repair, or insertion. This makes it a powerful approach for studying and potentially treating HCC [71]. Recent studies have shown that CRISPR-Cas9 can inhibit the expression of oncogenes in HCC cells, disrupt HBV replication by targeting viral DNA, and enhance the susceptibility of cancer cells to existing treatments [72]. In one of their studies, [PERSON] et al. investigated whether the CRISPR/Cas9 gene-editing system could be used to knock out the hepatitis B surface antigen (HBsAg) in HCC cells infected with HBV. Given that chronic HBV infection is a major contributor to liver cancer, the researchers aimed to determine whether eliminating HBsAg could reduce the cancerous behavior of these cells--specifically, their ability to proliferate and form tumors. UsingCRISPRR/Cas9 constructs designed to target the preS1/preS2/S open reading frame of the HBV genome, they successfully disabled the gene in HBV-positive HCC cell lines. The impact of the knockout was assessed through a series of in vitro assays, such as CCK-8-based proliferation tests and cell counting, which revealed a clear reduction in cell proliferation following HBsAgAg disruption. To evaluate tumorigenic potential, the team conducted both xenograft experiments in mice and immunohistochemical analyses. In both settings, cells lacking HBsAgAg showed a significantly reduced ability to form tumors. Overall, the study demonstrated that targeting HBsAgAg with CRISPR/Cas9 not only suppresses the growth of HBV-positive liver cancer cells but also diminishes their tumor-forming potential. These results suggest that this gene-editing approach could be a promising therapeutic strategy for HBV-related HCC. However, the study also presents some important limitations. Potential adverse effects on normal (non-tumor) hepatocytes were not discussed, limiting the safety assessment of the intervention. Additionally, the effect on cccDNA (covalently closed circular DNA)--the persistent form of HBV in hepatocytes--was not evaluated. This viral reservoir is notoriously difficult to eliminate and remains a major barrier to curing chronic HBV infection [73]. In another study, [PERSON] et al. explored the role of NSD1, a nuclear receptor-binding SET domain protein, in HCC progression. They used CRISPR/Cas9 to knock out NSD1 in HCC cell lines, aiming to disrupt tumor-related signaling--particularly the NSD1-H3-Wnt10b axis. After editing the NSD1 gene with specific guide RNAs, the team measured histone H3 methylation changes. The knockout increased H3K27 trimethylation and reduced H3K36 dimethylation, two modifications known to suppress gene transcription. These epigenetic changes blocked Wnt10b signaling, a key pathway in tumor growth. To evaluate the biological impact, the researchers performed cell proliferation assays and xenograft experiments in mice. NSD1-deficient cells showed slower proliferation, reduced tumor formation, and lower metastatic potential. These results indicate that NSD1 actively promotes HCC through both epigenetic regulation and oncogenic signaling, positioning it as a strong candidate for targeted liver cancer therapy. Although the results of this study are promising, it presents certain limitations, such as the use of a relatively small clinical sample size, the lack of functional validation of other pathways regulated by NSD1, and the absence of advanced in vivo models. Moreover, the risk of off-target effects associated with the use of CRISPR/Cas9 was not thoroughly evaluated, which could affect the accuracy of the conclusions [74].