A bacterial one-hybrid system to isolate homing endonuclease variants with altered DNA target specificities.

Chromosomal cleavage near the site of mutations that cause disease can facilitate the targeted repair of the locus. Gene therapy protocols therefore require the engineering of DNA endonucleases that target specific genomic loci. Here, we describe a bacterial one-hybrid selection system that has been used to isolate derivatives of the I-SceI homing endonuclease from combinatorial libraries that display altered DNA recognition specificities. The construction of plasmid expression libraries, the development of reporter strains, and the utilization of these components in the bacterial one-hybrid system are detailed.