Chembiochem : a European journal of chemical biologyJun 8, 2026

Improving Lipid Nanoparticles for More Effective Gene Silencing Using CRISPR/Cas9

mRNA Technology Weekly Brief ↗PubMed ↗DOI ↗

Updated Jun 9, 2026

Abstract

The optimized lipid nanoparticle formulation achieved >80% knockout of the luciferase reporter in HeLa-Luc cells.

Formulation optimization of lipid nanoparticles (LNPs) is crucial for enhancing the delivery of CRISPR/Cas9 gene-editing technologies.
High editing efficiency was observed alongside low cytotoxicity in selected LNP formulations.
Physicochemical properties, such as zeta potential and mean diameter around 200 nm, were associated with improved delivery performance.
Pairing superior cellular uptake and endosomal escape capabilities with optimal LNP compositions may synergistically enhance delivery efficiency.
Efficient protein-level knockdown of HSP47 was achieved in L929 cells using the optimized LNP to codeliver Cas9 mRNA and sgRNA.

Simplified

Efficient and safe delivery systems remain a major barrier to the clinical translation of CRISPR/Cas9 gene-editing technologies; among these, formulation optimization of lipid nanoparticles (LNPs) is a key approach to improve delivery performance. Here, we constructed an orthogonal formulation library of LNPs using the ionizable lipids 4A2C2C6-A8 and 4A2C2C8-A8 at varied molar ratios and screened for optimal compositions. We identified formulations that combined high editing efficiency with low cytotoxicity; in HeLa-Luc cells, the optimized formulation achieved >80% knockout of the luciferase reporter. Further physicochemical and functional investigations showed that LNPs with relatively high zeta potential, mean diameters near 200 nm, and appropriate internal hydrophobicity-when paired with superior cellular uptake and endosomal escape capabilities-synergistically enhanced delivery efficiency. Using the compositionally optimized LNP to codeliver Cas9 mRNA and an sgRNA targeting HSP47 (whose overexpression has been implicated in fibrosis), we achieved efficient protein-level knockdown of HSP47 in L929 cells. These results provide important guidance for formulation optimization of CRISPR/Cas9 LNPs and support their potential application in antifibrotic therapies.

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Improving Lipid Nanoparticles for More Effective Gene Silencing Using CRISPR/Cas9

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