Background: Genome editing technologies, including the Sleeping Beauty (SB) transposon system and CRISPR/Cas9 machinery, have caused a paradigm shift in both research and therapeutic applications. While widely utilized, there is a lack of direct comparative studies assessing their efficacy, specificity, and long-term performance, hindering the selection and optimization of these technologies for clinical translation. 

 

Methods: Transfection experiments were conducted in HeLa and HEK293 cells to compare the hyperactive SB transposase SB100X with CRISPR/Cas9 systems targeting ANTXR1, HBEGF, and PSEN2. For functional optimization of CRISPR/Cas9, a variety of sgRNA:donor ratios (1:1, 1:2, 2:1) were utilized. Efficacy was measured by quantification of transgene integration and the assessment of long-term expression via a colony-forming assay. Cell viability and toxicity were evaluated using a CCK-8 assay, while colony size, spatial distribution and morphology were analyzed using ImageJ. To assess the genome-wide specificity of the systems, a comparative analysis of existing NGS datasets was conducted.

 

Results: CRISPR/Cas9 demonstrated a clear short-term advantage, achieving ~10% higher transgene integration compared to SB100X, while also showing significantly higher transfection efficiency (~56%). The highest integration efficiency was observed at a 1:2 sgRNA:donor ratio. However, SB100X clearly outperformed CRISPR/Cas9 in long-term colony survival, resulting in 25-fold more colonies after selection. CRISPR/Cas9 colonies were more uniform, with higher circularity values and expressed lower immediate toxicity post-transfection. 

 

Conclusion: The results provide valuable insights into the applicability of both systems, suggesting that SB100X offers advantages for sustained gene expression and robust cell survival, while CRISPR/Cas9 may be more appropriate for precise gene editing.