Supplementary file 1_Overcoming delivery barriers of large all-in-one CRISPR/Cas9 plasmids using lysine-based lipid nanoparticles.docx

LNPs containing DOPE exhibited significantly higher transfection efficiency than those containing DOPC and were therefore selected for subsequent experiments. Next, LNPs formulated with different cationic or ionizable lipids were prepared and evaluated in HEK293 cells for transfection efficiency, cytotoxicity, and gene-editing efficiency.</p>Results<p>LNPs containing the commercial ionizable lipid SM-102 showed higher transfection and gene-editing efficiencies than the lysine-headgroup cationic lipids represent a promising and biocompatible platform for CRISPR/Cas9 plasmid delivery; however, increased cytotoxicity was observed in highly transfected cell populations.

In contrast, the lysine-headgroup cationic LMPs achieved effective delivery of large plasmid DNA and CRISPR/Cas9-mediated gene editing while maintaining superior biocompatibility.</p>Conclusion<p>These results demonstrate that lysine-headgroup cationic lipids represent a promising and biocompatible platform for CRISPR/Cas9 plasmid delivery. Further optimization of lipid composition and molar ratios may enhance transfection and gene-editing efficiencies, advancing the development of safer nonviral gene delivery systems.</p>