Nuclear-targeted CQDs-PP-mediated CRISPR/Cas9 Plasmid Delivery And Gene Editing

Nuclear targeted delivery has great potential in improving the efficiency of non viral vector mediated genome editing.However,there are still challenges in delivering CRISPR/Cas9 plasmid directly and effectively to the nucleus.In this study,a nuclear targeted delivery CRISPR/Cas9 system based on fluorescent carbon quantum dots(CQDs)was developed.Using citric acid as carbon source,polyethyleneimine(PEI)and polyethylene glycol(PEG)as passivators,the synthesis and passivation were carried out simultaneously,and CQDs-PEI-PEG(CQDs-PP)was synthesized by "one pot" hydrothermal method.Structural characterization and fluorescence analysis showed that CQDs-PP had the characteristics of ultra-small size(particle size 3 nm),positive surface charge and green emission fluorescence.DNA concentration experiments showed that CQDs-PP spontaneously and instantaneously formed nanocomposites with CRISPR/Cas9 plasmid through electrostatic attraction.CQDs-PP/p DNA nanocomposite transfected cells and fluorescence tracking showed that CQDs-PP was absorbed by cells through nest mediated endocytosis,directly entered the nucleus without lysosomal pathway,and released the plasmid in the nucleus.CQDs-PP carries CRISPR/Cas9 plasmid to transfect Hela cells.The transfection efficiency can be tracked by fluorescence to realize the insertion/deletion mutation of target gene EFHD1.CQDs-PP showed higher gene editing efficiency and equivalent or lower cytotoxicity than Lipo2000 and PEI passivated CQDs mediated transfection.Therefore,nuclear selectively located CQDs-PP is expected to be developed as an in vitro gene editing system for fluorescence tracking and nuclear targeted delivery of CRISPR/Cas9 carrier.