• Home
  • Search Results
  • Using defined finger-finger interfaces as units of assembly for constructing zinc-finger nucleases.

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

Nucleic acids research (2013-01-11)
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
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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Nde I from Neisseria denitrificans, Restriction Enzyme
Sigma-Aldrich
EclX I from Enterobacter cloacae 590, Restriction Enzyme

Social Media

LinkedIn icon
Twitter icon
Facebook Icon
Instagram Icon

MilliporeSigma

Research. Development. Production.

We are a leading supplier to the global Life Science industry with solutions and services for research, biotechnology development and production, and pharmaceutical drug therapy development and production.

© 2021 Merck KGaA, Darmstadt, Germany and/or its affiliates. All Rights Reserved.

Reproduction of any materials from the site is strictly forbidden without permission.