Homing Endonuclease Target Site Specificity Defined by Sequential Enrichment and Next-Generation Sequencing of Highly Complex Target Site Libraries

Homing endonucleases (HEs) are DNA sequence-specific enzymes that recognize and cleave long target sites (14–40 bp) to generate double-strand breaks (DSBs). Their high site recognition specificity and tight coupling of binding and cleavage make HEs attractive reagents for targeted genome manipulation. In order to delineate the target site specificity of HEs and facilitate HE engineering, we have developed a method for comprehensive target site profiling of HEs cleavage specificity using partially randomized target site libraries and high-throughput DNA sequencing.