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. 2015 Oct 14;9:355. doi: 10.3389/fnins.2015.00355

Figure 5.

Figure 5

Schematic representation of ZFN and TALEN-mediated homologous recombination into AAVS1 safe site of human chromosome. The FokI endonuclease enzyme derived from the prokaryote Flavobacteriumokeanokoites was found to function as two separate domains - one binds DNA in a sequence specific manner and one cleaves in a sequence independent manner that is highly specific because it needs dimerization for endonuclease activity on the target DNA. Therefore, by fusing a FokI monomer to two ZFPs or TALEs, which bind to adjacent sequences at a safe site on the human chromosome such as AAVS1 target site, it is possible to generate sequence specific DNA nuclease complexes that facilitate selective targeting and homologous recombination within human genomes (Davis and Stokoe, 2010). (1) Two ZFPs or TALEs are designed to specifically target AAVS1 site in anticipated clinically relevant target cell. These proteins are fused to a FokI cleavage domain to facilitate their nuclease activity. The ZFN or TALEN are co-delivered with the HR donor plasmid DNA vector into the cell by a non-viral gene delivery technique to reach the nuclei. (2) Inside the nucleus, ZFNs or TALENs recognize specific DNA sequences on the target cell's chromosome. (3) Binding of two ZFNs or TALENs complex to the target site allows FoKI to dimerize. (4) ZFNs or TALENs complex creates a targeted chromosome break at the AAVS1 site of the host cell, which facilitates HR between the donor DNA vector and homologous sequences. (5) After generating the DNA double strand break, the ZFNs or TALENs complex disassociate from target DNA. (6) The AAVS1 homology arms subcloned into the HR donor DNA vectors recognize the homolog sequences located on the host genome. (7) HR-mediated insertion of the therapeutic gene expression cargo occurs at the AAVS1 site of the clinically relevant host cell.