Using defined finger-finger interfaces as units of assembly for constructing zinc-finger nucleases

Cong Zhu, Ankit Gupta, Victoria L. Hall, Amy L. Rayla, Ryan G. Christensen, Benjamin Dake, Abirami Lakshmanan, Charlotte Kuperwasser, Gary D. Stormo, Scot A. Wolfe

Research output: Contribution to journalArticlepeer-review

26 Scopus citations


Zinc-finger nucleases (ZFNs) have been used for genome engineering in a wide variety of organisms; however, it remains challenging to design effective ZFNs for many genomic sequences using publicly available zinc-finger modules. This limitation is in part because of potential finger-finger incompatibility generated on assembly of modules into zinc-finger arrays (ZFAs). Herein, we describe the validation of a new set of two-finger modules that can be used for building ZFAs via conventional assembly methods or a new strategy-finger stitching-that increases the diversity of genomic sequences targetable by ZFNs. Instead of assembling ZFAs based on units of the zinc-finger structural domain, our finger stitching method uses units that span the finger-finger interface to ensure compatibility of neighbouring recognition helices. We tested this approach by generating and characterizing eight ZFAs, and we found their DNAbinding specificities reflected the specificities of the component modules used in their construction. Four pairs of ZFNs incorporating these ZFAs generated targeted lesions in vivo, demonstrating that stitching yields ZFAs with robust recognition properties.

Original languageEnglish
Pages (from-to)2455-2465
Number of pages11
JournalNucleic acids research
Issue number4
StatePublished - Feb 2013


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