UMMS Affiliation

Program in Gene Function and Expression; Department of Biochemistry and Molecular Pharmacology

Date

1-8-2013

Document Type

Article

Medical Subject Headings

Zinc Fingers

Disciplines

Biochemistry, Biophysics, and Structural Biology

Abstract

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 DNA-binding 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.

Comments

Citation: Nucleic Acids Res. 2013 Jan 8. Link to article on publisher's site

Copyright The Author(s) 2013. Published by Oxford University Press.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Related Resources

Link to Article in PubMed

PubMed ID

23303772

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