An optimised lipid nanoparticle platform enables efficient CRISPR/Cas9 genome editing in hard-to-transfect cells

CRISPR/Cas9 is a powerful tool for genome editing and functional gene studies, but its therapeutic potential is often hampered by inefficient transfection, particularly in hard-to-modify cell types. In this study, we developed and optimised a lipid nanoparticles (LNPs) platform that enhances CRISPR/Cas9-mediated genome editing across diverse cell types, including those that are difficult to modify using commercially available lipid-based delivery agents. Our engineered LNPs exhibit consistent particle size below 100 nm, low polydispersity and high encapsulation efficiency. Using this platform, GFP knockout in HEK-293 cells reached 78.7%, and maintained consistent efficiency after lyophilisation and reconstitution. Knockout of the LCN2 gene in MDA-MB-231 cells resulted in a 90.8% reduction in mRNA expression, outperforming the 51.1% reduction achieved using CRISPRMAX Lipofectamine, and functional assays confirmed that LCN2 disruption significantly inhibited cell proliferation and migration. Co-delivery of CRISPR/Cas9 and a GFP HDR template enabled precise knock-in, achieving >20% efficiency in HEK-293 cells and >8% in MSCs and DC2.4 cells, significantly outperforming Lipofectamine 3000 transfection reagent. Given the rapid expansion of CRISPR applications in biomedical research, our LNP-based delivery system represents a promising non-viral platform with broad potential for therapeutic applications, particularly in hard-to-modify cell types. Statement of Significance CRISPR/Cas9 is a transformative gene-editing technology for functional genomics and therapeutic development; however, its widespread application is constrained by the lack of safe and efficient delivery systems, particularly for hard-to-transfect cell types. Lipid nanoparticles (LNPs) represent a promising non-viral delivery strategy, yet their transfection efficiency varies substantially across cell lines due to differences in cellular membrane properties. Here, we developed and optimized an LNP platform that enables highly efficient Cas9/sgRNA-mediated gene knockout and knock-in across multiple challenging cell types, consistently outperforming commercial transfection reagents. The findings establish this LNP system as a versatile and effective non-viral platform for CRISPR/Cas9-mediated genome editing, overcoming key limitations of existing commercial lipid-based delivery agents in hard-to-modify cell types and offering a promising strategy for future clinical translation.