Correction Of Tyrosinemia Type 1 In Adult Rats Through Viral Delivery Of CRISPR/Cas9 System

With the concept of Precision Medicine being put forward and implement,many biological researchers urgently need an efficient method in order to studying genes function more efficiently and accurately,thus we can better understand the pathogenesis of disease and achieve the goal of Individual Precision Medical Treatment.Recent years,new gene editing technologies are constantly emerging,especially the development and application of CRISPR/Cas(clustered regularly interspaced short palindromic repeats/CRISPR associated proteins)system,which provided an efficient and convenient method to scientists who can achieve the purpose of gene site-specific modification.Among the several CRISPR/Cas9 systems,the type II CRISPR/Cas is the most widely used system.Currently,the CRISPR/Cas9 system has been used for efficient editing of single gene or multiple genes in different cells and tissue,this technique shows a broad application prospect.Therefore,treat disease by using gene editing techniques become the foundation and important research aspect of Precision Medical Treatment.However,how to import the CRISPR/Cas9 and template of gene repair efficiently and safely,which become a key challenge for gene therapy.Compare with mouse,the rat is more similar with human in physiology,ethology and toxicology.What's more,there are still few researches on gene editing in adult rats,therefore it makes sense to achieve precise gene repair in adult rats.Besides,the CRISPR/Cas9 technique also has a problem of off-target,and how to reduce the off-target rate become the key issue of CRISPR/Cas9 furture application.In this study,we delivered different variants of Cas9 protein(Cas9/Cas9n(Cas9 nickase))and the Rapamycin induced Cas9(Split-Cas9)into hepatic cell of adult rats via adenovirus and adeno-associated virus(AAV)system.As the result,we have not only achieved the gene repair at specific sites in liver of adult rat successfully,but reduced the off-target rate significantly,our work can lays a solid foundation for the clinical application of gene therapy.In this study,we established a FAH rat model,which can fine simulate the human hereditary tyrosinemia type 1(HTI).Specifically,we injected the Cas9 mRNA and sgRNA that target rat Fah gene into one-cell stage rat embryos,and then get the model rat with Fah deficiency.Second,we amplified and purified the adenovirus packed with Cas9/Cas9n and gene repair template.These adenovirus affected the liver of rat and achieved gene editing successfully after tail vein injection of adenovirus.Considering the safety of clinical application,we packed the separated,Rapamycin induced Split-Cas9 system and gene repair template in AAVs at the same time.In this way,we also have achieved the gene repair at specific sites in adult rat.In summary,our study achieved gene accurate repair in adult rat by using multiple methods in different aspect,these results can provide scientific bases for clinical application of CRISPR/Cas9 systems,and further promote the development of Precision Medicine.