Journal
NATURE PROTOCOLS
Volume 4, Issue 10, Pages 1471-1501Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/nprot.2009.98
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Funding
- National Institutes of Health [R01 GM069906, R24 GM078369, R21 RR024189, R33 GM066387]
- MGH Pathology Service
- National Science Foundation [DBI 0501678]
- Pioneer-Hi-Bred International 2008 Graduate Fellowship
- Iowa State University CIAG
- NATIONAL CENTER FOR RESEARCH RESOURCES [R21RR024189] Funding Source: NIH RePORTER
- NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES [R01GM069906, R24GM078369, R21GM066387, R33GM066387] Funding Source: NIH RePORTER
- OFFICE OF THE DIRECTOR, NATIONAL INSTITUTES OF HEALTH [DP1OD006862] Funding Source: NIH RePORTER
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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 similar to 8 weeks.
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