A Novel CRISPR/Cas9 Gene Editing System Delivered By Multifunctional Chimaeric Peptide

CRISPR/Cas9-based gene editing therapy that directly manipulates diseaserelated genes in genomes,have a potential to revolutionize the gene therapy.Compared with ex vivo therapy,gene editing therapy in vivo is more challenging as for their higher requirements for the efficiency,safety,and accuracy of the delivery system.Cas9/sgRNA is assembled in vitro to form ribonucleoprotein(RNP),which is the safest form for CRISPR.However,lipid nanoparticles and adeno-associated virus vectors,which was widely utilized in clinical trials,cannot achieve the delivery of RNP in vivo,which limits the development of CRISPR/Cas9-based gene editing therapy.A novel multifunctional chimeric peptide-based biomacromolecule delivery system eTAT(enhanced TAT)was provided by our research group previously,showing the efficient endosomal escape and high serum tolerance.It has been demonstrated that eTAT system could mediate intracellular delivery of protein macromolecules in vitro and in vivo.In this study,we found that eTAT could mediate intracellular uptake of protein-nucleic acid complexes as well.Correspondingly,we aimed to establish a novel CRISPR/Cas9 RNP gene editing system delivered by chimeric peptide eTAT,and evaluated its feasibility of in vivo gene editing and its potential in disease therapy.This study mainly includes two parts:the knock-out gene editing system and the homology repair gene editing system.The first part in this study is about the establishment and application of the CRISPR/Cas9 gene knock-out system based on eTAT-Cas9 RNP.The system can induce a knockout efficiency of about 70%of the BFP at the cellular level,which is better than the commercial liposome,CRISPR MAX.Moreover,it exhibits the advantages of fast-editing,low off-target effect,and no cell selectivity.eTAT-Cas9 RNP mediated knock-out of the PD-L1 and Plk1 genes in the mouse model inhibits the growth of malignant tumors,confirming its potential of gene editing therapy in vivo.The second part in this study focus on establishment and application of the CRISPR/Cas9-mediated homology repair system.It is more challenging to develop homology-directed repair(HDR)-based gene editing as for requiring the simultaneous delivery of Cas9 RNP and donor DNA in vivo.For this reason,we constructed a novel donor DNA delivery system based on eTAT fused-DNA specific binding protein,and its combination with eTAT-Cas9 RNP induces efficient gene correction and gene insertion.Then,all HDR elements are integrated in one complex,coined All-in-One HDR complex.This complex is successfully applied in the treatment of hereditary tyrosinemia in mice,but also shows no obvious toxicity during the treatment.In general,the establishment of the CRISPR/Cas9 gene editing system based on chimeric peptides,is a novel approach for gene editing in vivo,and will be one of the important candidate molecules for gene editing therapy as well.