Genome Editing Is Facilitated By Simultaneous Delivery Of SgRNA And Donor DNA In A Single Adeno-associated Virus And CRISPR/Cas9-expressing Adenovirus

Objective: To construct the Adeno-associated virus(AAV) expression vector pAAVeG-U6gRNA and verify its gene editing function by creating different gene editing models according to different receptor cells.Methods: First, to reconstruct the primary Eukaryotic expressing vector phU6-gRNA into AAV vector pAAVeG-U6gRNA expressing both gRNA and Donor DNA through gene cloning method. After that, we confirmed the vector sequence by DNA sequencing. Then we packaged the new AAV vector into virus using AAV-DJ Helper Free Bicistronic System, and infected the muscle cells with packaged virus particles to verify its function of infection. Second,three gene editing models have been created to verify the function of vector pAAVeG-U6gRNA, including Slug deletion model, Mdx correction model and Slug-tag insertion model. We then designed specific sgRNA and donor DNA according each model and cloned them into AAV vector pAAVeG-U6gRNA.After that, we packaged the AAV vector expressing sgRNA and donor DNA into adeno-associated virus(AAV) in 293T cells with AAV-DJ Helper Free Bicistronic Expression System. Then, we co-infected the receptor cells with packaged AAV and CRSIPR/Cas9-expressing adenovirus. Finally, experimental technique of PCR, Western Blot, drug selection and gene sequencing were used to test the function of AAV vector pAAVeG-U6gRNA in gene deletion,insertion and correction models.Results: Receptor cells infected with AAV and CRISPR/Cas9-expressing adeno-virus expressed green fluorescence and red fluorescence indicated successfully infection. 48 hours after infection, infected receptor cells were observed with inverted fluorescence microscope, the infection rates were 70%.Results of PCR, gene sequencing and Western Blot showed the new constructed AAV vector play a good role in CRISPR/Cas9 mediated gene editing.Conclusion: AAV vector pAAVeG-U6gRNA co-expressing sgRNA and donor DNA has been successfully constructed.