Clinical application for precisely modifying the human genome with engineered zinc finger nuclease ZFN technology is largely hindered by cytotoxicity. Engineered zinc-finger nucleases (ZFNs) cut a DNA double-strand at a predetermined target site in the human genome for stimulating gene repair via homologous recombination (Hadel, Alwin, & Cathomen, 2009). Cytotoxicity is proposed to be caused by cleavage at “off-target sites” (Miller et al., 2007), (Cathomen & Joung, 2008), (Cornu et al., 2007), (Carroll, 2008), However, the phenomenon of cytotoxicity is not well understood, because of a fundamental lack of understanding of even simple DNA – protein interactions and the existence of unknown target sites a selected ZFN might bind specifically.

Though a large number of zinc finger libraries (Ding, Lorenz, Kreutzer, Li, & Thiesen, 2009; Fu et al., 2009) (Blancafort, Segal, & Barbas III, 2004), (Bae, Do Kwon, Shin, Hwang, & Ryu, 2003) have been reported, detailed understanding of the transcription factor binding sites (Tompa, 2005) p.144 and DNA-protein interactions (Desai, Rodionov, Gelfand, Alm, & Rao, 2009) p. 2500 have not been fully addressed. This resulted in high failure rates of modular assembly of engineered zinc finger arrays (Ramirez et al., 2008) p. 374 and cytotoxicity in human cells which is thought to be caused by cleavage at “off-target” sites (Cornu et al., 2007).

The successful clinical use of modular assembled zinc finger arrays for clinical application rests on the requirement that the arrays only recognize and edit the genome at one precise location which is the target location or target binding site. However, it has been reported that modular assembled zinc finger arrays also interfere and modify the genome at unspecified locations (Cathomen & Joung, 2008), (Cornu et al., 2007). Those unspecified locations (these are locations that are unknown to the researcher) have been referred to as “off-target” locations or binding sites.

In this project we identified the actual number of the Sp1 binding site GGGGCGGGG on the entire Human Genome.


Cytotoxicity 1

Cytotoxicity 2

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