LNP-mediated CRISPR gene editing of human vascular endothelial cells in 2D and 3D microfluidic cell culture systems

Efficient and cell-type-specific delivery remains a major barrier to CRISPR-based genome editing in vascular endothelial cells. Here, we systematically evaluate lipid nanoparticle (LNP)-mediated mRNA and CRISPR delivery in human umbilical vein endothelial cells (HUVECs). While a conventional SM-102-based LNP formulation enables highly efficient mRNA delivery to both HEK293T cells and HUVECs, CRISPR-SpCas9 gene editing is observed exclusively in HEK293T cells, revealing strong cell-dependent limitations. We identify reduced SpCas9 mRNA translation as a key bottleneck in endothelial cells and demonstrate that rational modification of LNP lipid composition dramatically enhances SpCas9 protein expression and restores robust genome editing in HUVECs. Using the optimized LNP formulation, we achieve efficient editing of VEGFA and HIF1A in both 2D cultures and physiologically relevant 3D microfluidic endothelial systems mimicking flow conditions in vivo. Together, our results establish lipid composition as a critical determinant of endothelial CRISPR editing efficiency and provide a scalable, non-viral strategy for genome engineering in the vascular endothelium.