Quantifying the information content of homing endonuclease target sites by single base pair profiling

Homing endonucleases (HEs) are native proteins that recognize long DNA sequences with high site specificity in vitro and in vivo. The target site specificity of HEs is high, though not absolute. For example, members of the well-characterized LAGLIDADG family of homing endonucleases (the LHEs) recognize target sites of ~20 base pairs, and can tolerate some target site base pair changes without losing site binding or cleavage activity. This modest degree of target site degeneracy is practically useful once defined and can facilitate the engineering of LHE variants with new DNA recognition specificities. In this chapter, we outline general protocols for systematically profiling HE target site base pair positions in order to define their functional importance in vitro and in vivo, and show how information theory can be used to make sense of the resulting data.