Identification and Analysis of Genomic Homing Endonuclease Target Sites

Homing endonucleases (HEs) are highly site-specific enzymes that enable genome engineering by introducing DNA double-strand breaks (DSB) in genomic target sites. DSB repair from an HE-induced DSB can promote target site gene deletion, mutation, or gene addition, depending on the experimental protocol. In this chapter we outline how to identify potential genomic target sites for HEs with known target site specificities and the different experimental strategies that can be used to assess site cleavage in living cells. As an example of this approach, we identify potential human genomic target sites for the LAGLIDADG HE I-CreI that, by nine different selection criteria, may be new “safe harbor” sites for gene insertion.