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Hum Gene Ther Methods 2018 Jun;29(3):135-45

Efficiency and specificity of targeted integration mediated by the AAV2 Rep 78 protein.

Li P, Marino MP, Zou J, Argaw T, Morreale MT, Iaffaldano BJ, Reiser J


The 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, we tested plasmid-based donor vectors 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. We also compared the Rep 78 and ZFN-based approaches in HEK 293 cells and in human induced pluripotent stem cells using integrase deficient lentiviral vectors. The targeting efficiencies involving the Rep 78 protein were similar to those involving the AAVS1-specific ZFN, while the targeting specificity for the Rep 78 protein was lower compared to that of the ZFN. We anticipate that the Rep 78 nickase-based targeting approach may ultimately contribute to the reduction of risks associated with other genome editing approaches involving DNA double strand breaks.

Category: Journal Article
PubMed ID: #29860898 DOI: 10.1089/hgtb.2018.052
PubMed Central ID: #PMC6016731
Includes FDA Authors from Scientific Area(s): Biologics
Entry Created: 2018-06-03 Entry Last Modified: 2018-07-09