ArticleFrontiers in genome editing2026
Overcoming delivery barriers of large all-in-one CRISPR/Cas9 plasmids using lysine-based lipid nanoparticles.
Article in Frontiers in genome editing, 2026. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Not yet cited in PubMed.
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Abstract
Introduction: Lipid nanoparticles (LNPs) are widely used as drug delivery systems (DDS) for the intracellular delivery of gene-editing tools such as the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system. Previously, a lysine-headgroup cationic lipid (K3C16) was reported to exhibit high biocompatibility and cellular internalization via plasma membrane fusion when formulated as liposomes, suggesting its potential as a low-toxicity gene delivery material. However, the translation of this lipid into a multi-component lipid nanoparticle (LNP) matrix via microfluidic engineering for large cargo encapsulation has not been explored. In this study, K3C16 was successfully engineered for the first time as the main lipid component of LNPs to deliver large plasmid DNA encoding the CRISPR/Cas9 system. Methods: First, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) were evaluated as helper phospholipids to identify an optimal lipid composition. 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. Results: 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. Conclusion: 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.
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