Standardized reagents and protocols for engineering zinc finger nucleases by modular assembly.
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.
Wright
David A
DA
Department of Genetics, Development & Cell Biology, Iowa State University, 1035A Roy J. Carver Co-Lab, Ames, Iowa 50011, USA.
Thibodeau-Beganny
Stacey
S
Sander
Jeffry D
JD
Winfrey
Ronnie J
RJ
Hirsh
Andrew S
AS
Eichtinger
Magdalena
M
Fu
Fengli
F
Porteus
Matthew H
MH
Dobbs
Drena
D
Voytas
Daniel F
DF
Joung
J Keith
JK
D001419:Bacteria / Q000235:genetics; D019295:Computational Biology / Q000379:methods*; D003851:Deoxyribonucleases / Q000138:chemical synthesis* / Q000737:chemistry; D005818:Genetic Engineering / Q000379:methods*; D010957:Plasmids / Q000235:genetics; D012984:Software; D020798:Two-Hybrid System Techniques; D016335:Zinc Fingers / Q000235:genetics*
D003851:Deoxyribonucleases