Rapid determination of homing endonuclease DNA binding specificity profile

Evaluating the binding specificity and identifying the most preferred target sequence for a homing endonuclease often represents a key step during its characterization, engineering, and application for genome engineering. This chapter describes a high-throughput, fluorescence-based, competition-binding assay which can be used to measure the relative binding affinities of the homing endonuclease to a large number of DNA target site variants in a single experiment. The base preference at each position of the target sequence can be quantitated based on this assay, and the overall binding specificity of the enzyme can thereby be determined and compared with its cleavage specificity.