MiRNA Responsive Cas9/sgRNA Nano Delivery System For Cytoplasmic Proteins Direct Delivery And Enhanced Genome Editing

Nanocarrier system based on Cas9/sgRNA complex direct delivery hold great significance for CRISPR-based systems and precise treatment of multiple genetic disorder diseases.Herein,the master's thesis work,we developed a miRNA-responsive Cas9/sgRNA complex nano-delivery platform based on nanocarrier for gene editing system of specific targeting tumor cells,achieve direct cytoplasmic of Cas9/sgRNA complex efficient delivery and enhanced gene editing effect.Highly biocompatible DNA nano-vehicles can efficiently load Cas9/sgRNA complex by hybridization to sgRNA sequence.More Importantly,through programmable sequence design and fulcrum-based toehold-mediated chain replacement process,The Cas9/sgRNA complex on nanoflowers can be replaced by miRNAs specifically overexpressed by tumor cells,thereby achieving tumor cell-specific release of Cas9/sgRNA.Based on the idea,we developed a DNA nanoflower(DNF)based miR-21 respo nsive Cas9/sgRNA complex delivery system.DNA nanoflowers were synthesized by rolling circle amplification have characteristics of programmability,good biocompati bility and degradability under physiological conditions,etc.Due to its easy-to-progra m sequence design,we designed a nucleic acid aptamer on the nanoflower sequence s tructure that specifically targets the MUC1 protein,the achieved that specific targetin g of nanocarrier to tumor cells.By designing repetitive sequences that could hybridiz e miR-21,combined with the sgRNA of stem-loop structure modification(The stem-lo op structure has the same sequence as the miR-21 part),to load Cas9/sgRNA on DN F by sequence hybridization,Assembly into DNF/Cas9/sgRNA nanoformulations.In t he actual delivery process,the nanoformulation enters the lysosome inside the cell thr ough MUC1-mediated endocytosis,The endosomal/lysosomal escape induced by the massive release of Mg2+caused by the disintegration of nanoflowers under acidic con ditions directly delivers the Cas9/sgRNA complex to the cytoplasm.Subsequently,mi R-21 in the cytoplasm will replace the Cas9/sgRNA complex bound to DNF through a toehold-mediated sequence replacement reaction.owing to the nuclear-localization-signal(NLS)peptides fused to Cas9 protein,the released Cas9/sgRNA complex can e fficiently enter the nucleus,and by targeting the coding region of a specific gene,gen e editing is performed to suppress the expression of related genes.We have verified in cell experiments and animal experiments that DNA nanoflower-based miRNA-responsive nanocarriers could significantly improve the efficiency of Cas9/sgRNA complex delivery for gene editing compared with non-responsive release control group.This strategy is proposed to for the design of stimulus-responsive Cas9/sgRNA nano-delivery system and improve the gene editing effect of CRISPR system in vivoprovide new ideas and methods.