Background: The CRISPR-Cas9 system, a powerful tool, has revolutionized genome engineering in eukaryotic cells and living organisms. However, this approach poses unique concerns and limitations when used by conventional transfection methods, including limited packaging size and low delivery efficiency. Here, we aim at assessing the transfection efficiency of DNA encoding for the CRISPR-Cas9 system by PEI coated Magnetic NanoParticle (MNPs) to improve the delivery of CRISPR/Cas9 constructs into eukaryotic cells.
Results: Superparamagnetic iron oxide nanoparticles (SPIONs) coated with polyethylenimine (PEI) and then complexed with pCXLE-dCas9VPH-T2A-GFP-shP53 plasmid DNA. We used HEK-293 (human embryonic kidney) and Human foreskin fibroblasts (HFF) cells to express GFP after transfection to evaluate delivery efficiency with MNPs and Lipofection methods. PEI-coated nanoparticles with magnetic iron oxide core were synthesized by co-precipitation technique resulting in an average size of ~ 20 nm in diameter. Characterization of Magnetic Nano Particle (MNPs) revealed that particles have narrow size distribution sufficient colloidal stability. The result showed that the magnetofection method with an efficiency around 85.7% for HEK-293 and 28.2% for HFF. Also, transfection efficiency by lipofection method was 83.2% and 7.89% for HEK-293 and HFF respectively.
Conclusion: The magnetofection was revealed to be more efficient than classic Lipofectamine transfection as measured by GFP expression. We show that PEI-MNPs enable effective delivery and improved safety of plasmids encoding CRISPR/Cas9 into eukaryote cells.