Oligomerized pool engineering (OPEN): an 'open-source' protocol for making customized zinc-finger arrays.
Engineered zinc-finger nucleases (ZFNs) form the basis of a broadly applicable method for targeted, efficient modification of eukaryotic genomes. In recent work, we described OPEN (oligomerized pool engineering), an 'open-source,' combinatorial selection-based method for engineering zinc-finger arrays that function well as ZFNs. We have also shown in direct comparisons that the OPEN method has a higher success rate than previously described 'modular-assembly' methods for engineering ZFNs. OPEN selections are carried out in Escherichia coli using a bacterial two-hybrid system and do not require specialized equipment. Here we provide a detailed protocol for carrying out OPEN to engineer zinc-finger arrays that have a high probability of functioning as ZFNs. Using OPEN, researchers can generate multiple, customized ZFNs in approximately 8 weeks.
Maeder
Morgan L
ML
Molecular Pathology Unit, Center for Cancer Research, and Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, Massachusetts, USA.
Thibodeau-Beganny
Stacey
S
Sander
Jeffry D
JD
Voytas
Daniel F
DF
Joung
J Keith
JK
D001431:Bacteriological Techniques; D001435:Bacteriophages / Q000235:genetics; D001665:Binding Sites; D004926:Escherichia coli / Q000235:genetics; D015723:Gene Library; D023281:Genomics / Q000379:methods; D016133:Polymerase Chain Reaction; D015202:Protein Engineering / Q000379:methods*; D011995:Recombination, Genetic; D012984:Software; D016335:Zinc Fingers