Standardized reagents and protocols for engineering zinc finger nucleases by modular assembly

David A. Wright, Stacey Thibodeau-Beganny, Jeffry D. Sander, Ronnie J. Winfrey, Andrew S. Hirsh, Magdalena Eichtinger, Fengli Fu, Matthew H. Porteus, Drena Dobbs, Daniel F. Voytas, J. Keith Joung

Research output: Contribution to journalArticlepeer-review

147 Scopus citations

Abstract

Engineered zinc finger nucleases can stimulate gene targeting at specific genomic loci in insect, plant and human cells. Although several platforms for constructing artificial zinc finger arrays using "modular assembly" have been described, standardized reagents and protocols that permit rapid, cross-platform "mixing-and-matching" of the various zinc finger modules are not available. Here we describe a comprehensive, publicly available archive of plasmids encoding more than 140 well-characterized zinc finger modules together with complementary web-based software (termed ZiFiT) for identifying potential zinc finger target sites in a gene of interest. Our reagents have been standardized on a single platform, enabling facile mixing-and-matching of modules and transfer of assembled arrays to expression vectors without the need for specialized knowledge of zinc finger sequences or complicated oligonucleotide design. We also describe a bacterial cell-based reporter assay for rapidly screening the DNA-binding activities of assembled multi-finger arrays. This protocol can be completed in approximately 24-26 d.

Original languageEnglish (US)
Pages (from-to)1637-1652
Number of pages16
JournalNature Protocols
Volume1
Issue number3
DOIs
StatePublished - Aug 2006

Fingerprint Dive into the research topics of 'Standardized reagents and protocols for engineering zinc finger nucleases by modular assembly'. Together they form a unique fingerprint.

Cite this