Efficiency and Specificity of Targeted Integration Mediated by the Adeno-Associated Virus Serotype 2 Rep 78 Protein.

The adeno-associated virus serotype 2 (AAV2) Rep 78 protein, a strand-specific endonuclease (nickase) promotes site-specific integration of transgene sequences bearing homology arms corresponding to the AAVS1 safe harbor locus. To investigate the efficiency and specificity of this approach, plasmid-based donor vectors were tested in concert with nuclease encoding vectors, including an engineered version of the AAV2 Rep 78 protein, an AAVS1-specific zinc finger nuclease (ZFN), and the CRISPR-Cas9 components in HEK 293 cells. The Rep 78 and ZFN-based approaches were also compared in HEK 293 cells and in human induced pluripotent stem cells using integrase deficient lentiviral vectors.