Journal
NATURE METHODS
Volume 8, Issue 9, Pages 765-U115Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/nmeth.1670
Keywords
-
Categories
Funding
- US National Institutes of Health (NIH) [R01 GM065400]
- Defense Advanced Research Projects Agency [HR0011-11-2-0003]
- Howard Hughes Medical Institute
- NIH [R01 GM088040, DP1 OD006862, P30 NS045776]
- Jim and Ann Orr Massachusetts General Hospital
- National Science Foundation
- Ford Foundation
Ask authors/readers for more resources
Engineered zinc-finger nucleases (ZFNs) are promising tools for genome manipulation, and determining off-target cleavage sites of these enzymes is of great interest. We developed an in vitro selection method that interrogates 10(11) DNA sequences for cleavage by active, dimeric ZFNs. The method revealed hundreds of thousands of DNA sequences, some present in the human genome, that can be cleaved in vitro by two ZFNs: CCR5-224 and VF2468, which target the endogenous human CCR5 and VEGFA genes, respectively. Analysis of identified sites in one cultured human cell line revealed CCR5-224-induced changes at nine off-target loci, though this remains to be tested in other relevant cell types. Similarly, we observed 31 off-target sites cleaved by VF2468 in cultured human cells. Our findings establish an energy compensation model of ZFN specificity in which excess binding energy contributes to off-target ZFN cleavage and suggest strategies for the improvement of future ZFN design.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available