Gene Editing In Adult Mice Via Viral Delivery Of CRISPR/Cas9 System

The emerging CRISPR/Cas system has been broadly accepted as a revolutionary technology to engineer genomic DNA with high efficiency and accuracy.Pioneer studies suggest that CRISPR/Cas9 system can be used In gene therapy through site-specific correction of mutant gene in special disease models in vivo.The most critical step of therapeutic genome editing is delivery of CRISPR/Cas9 system in vivo.In this study,we have set up systems to produce high titer recombinant lentivirus,adenovirus and adeno-associate virus to delivery Cas9/sgRNA system for in vivo geome editing in mouse liver.First,by optimizing the lentivirus packaging system,we obtained the CRISPR/Cas lentivirus which titer reached 7.9×1010 IU/mL and achieved 5.21%genome editing efficiency of the F9 gene in murine liver within one month following intravenous injection of the CRISPR/Cas lentivirus.Due to the low genome modification efficiency and integration risk of lentivirus,we then optimized the adenovirus amplification and purification system,and obtained the adenovirus with titer reaching 7.5×1012 PFU/mL.The purified adenovirus infected murine liver cells successfully,with 100%infection rate and achieved 18.9%genome editing efficiency.The adeno-associated virus is currently the most widely used gene therapy vector with high efficiency and low toxicity.So we also optimized AAV packaging system.We obtained the AAV titering 2×1013 vg/mL,which can infect liver cells and cardiac muscle cells in vivo with 80%infection rate.This project optimized three high titer virus platforms,which provided the efficient delivery vehicles for CRISPR/Cas9 system and will be used to study the feasiability of CRISPR/Cas9 mediated gene therapy of more disease models in vivo.